Universal chloroplast integration and expression vectors, transformed plants and products thereof

ABSTRACT

The invention provides universal chloroplast integration and expression vectors which are competent to stably transform and integrate genes of interest into chloroplast genome of multiple species of plants. Transformed plants and their progeny are provided. Monocotyledonous and dicotyledonous plants are transformed which have never been transformed heretofore. Plants transformed with a synthetic gene express valuable biodegradable protein-based polymers (PBPs). Transformed plants produce high value molecules. Resistance is provided to agricultural crops against the major classes of chemical herbicides. Herbicide resistance is used as a lethal selectable marker for chloroplast transformation. The transformed plants are capable of expressing in addition to the targeted trait, a desirable, secondary non-targeted trait. Insect resistance is provided to transformed plants, both against insects that are susceptible to Bt toxins and against insects that have developed resistance to Bt toxins.

RELATED APPLICATIONS

This application is a continuation of U.S. Ser. No. 09/079,640 filed May15, 1998, now U.S. Pat. No. 7,129,391, which claims benefit ofprovisional application Ser. No. 60/079,042 filed Mar. 23, 1998, andprovisional application No. 60/055,314 filed Aug. 7, 1997, bothincorporated herein by reference in their entirety.

FIELD OF THE INVENTION

This application pertains to the field of genetic engineering of plantgenomes, particularly the genetic engineering of the genome of plantplastids, such as chloroplasts and to the stable transformation ofchloroplast genome of any plant species.

RELATED CASES

This application relates in particular to a universal chloroplastexpression and integration vector which is competent to transform anyplant with one or more genes of interest. The earlier U.S. Pat. No.5,932,479 teaches plant cells transformed by means of an expressioncassette comprising an exogenous DNA sequence which is stably integrated(covalently linked) to the chloroplast genome of the cell of a targetplant. “Stably” integrated DNA sequences are those which are inheritedthrough genome replication by daughter cells or organisms. Thisstability is exhibited by the ability to establish permanent cell lines,clones, or transgenic plants comprised of a population containing theexogenous DNA.

Likewise, U.S. Pat. No. 5,693,507 (1997) to Daniell and McFaddendiscloses such stable integration by means of an expression cassettewhich comprises an exogenous DNA sequence which codes for a desiredtrait, and the transformed plants.

BACKGROUND OF THE INVENTION

Advantages of Chloroplast Transformation Over Nuclear Transformation.The attractiveness of transformation of the chloroplast genome overtransformation of the nuclear genome is attributable to the seriousrisks resulting from the latter. One common concern is the escape offoreign genes through pollen dispersal from transgenic crop plants totheir weedy relatives. It has been demonstrated that transgenic pollenwill deliver foreign (transgenic) genes to other sexually-compatibleplants (detected by marker gene prevalence in progeny harvested fromnon-transgenic plants grown in surrounding area). For Example, dispersalof pollen from a central test plot containing transgenic cotton plantsto surrounding non-transgenic plants has been observed at varyingdistances in different directions. (Lewellyn and Fitt, 1996); (Umbeck,P. F., et al., 1991). In addition, the frequencies of marker genes inwild sunflowers averaged about 28 to 38%; in wild strawberries growingwithin 50 meters of a strawberry field, more than 50% of the wild plantscontained marker genes from cultivated strawberries. (King, J., 1996).

The escape of foreign genes through pollen is especially a seriousenvironmental concern, in the case of herbicide resistance genes,because of the high rates of gene flow from crops to wild relatives. Theconcern is that gene escape from transgenic crops to their weedyrelatives will create super weeds. In rice (Oryza sativa), gene flowfrom cultivated varieties to wild relatives has been noted, into O.perennis (Barrett, 1983) and red rice (O. sativa; Langevin et al.,1990). In the southern US, red rice has become a major weed becauseherbicides that kill it also kills cultivated rice. Decreased prices arepaid for cultivated rice contaminated with red rice. Some researchershave introduced the bar gene conferring resistance to glufosinate(Liberty) into cultivated rice to combat this weed (Oard et al., 1996;Sankula et al., 1996). However, due to sexual compatibility,introduction of a nuclear-expressed gene will allow transmission of thatresistance trait into red rice via pollen.

Similarly, transgenic oil seed rape, genetically engineered forherbicide resistance outcrossed with a weedy relative, Brassicacampestris (field mustard) and conferred herbicide resistance even inthe first back-cross generation under field conditions. (Mikkelson, T.R., et al., 1996).

Maternal inheritance of introduced genes prevents gene escape throughpollen. Engineering foreign genes through chloroplast genomes (which arematernally inherited for most of the crops) is a solution to thisproblem. Also, the target enzymes or proteins for most herbicides (e.g.amino acid/fatty acid biosynthetic pathways or photosynthesis) arecompartmentalized within the chloroplast. Another important advantage ofchloroplast transformation is the higher levels of foreign geneexpression due to a very high copy number (5000-10,000) of chloroplastgenomes in plant cells. Because the transcriptional and translationalmachinery of the chloroplast is prokaryotic in nature, herbicideresistant genes of bacterial origin can be expressed at extraordinarilyhigh levels in chloroplasts.

Transformation of the Chloroplast Genome. Early investigations onchloroplast transformation focused on the development of in organellosystems using intact chloroplasts capable of efficient and prolongedtranscription and translation (Daniell and Rebeiz, 1982; Daniell et al.,1983) and expression of foreign genes in isolated chloroplasts (Danielland McFadden, 1987). These experiments were done under the premise thatit was possible to introduce isolated intact chloroplasts intoprotoplasts and regenerate transgenic plants (Carlson, 1973). Thediscovery of the gene gun as a transformation device opened thepossibility of direct plastid transformation in plants (Daniell, 1993).Transient expression of foreign genes in plastids of dicots (Daniell etal., 1990; Ye et al., 1990), monocots (Daniell et al., 1991), prolongedforeign gene expression using autonomously replicating chloroplastexpression vectors (Daniell et al., 1990) and stable integration of aselectable marker into the tobacco chloroplast genome (Svab and Maliga,1993) were accomplished using the gene gun. Tobacco plants resistant tocertain insects were obtained by integrating the cryIAc gene into thetobacco chloroplast genome (McBride et al., 1995; U.S. Pat. No.5,451,513, incorporated herein by reference). Stable plastidtransformation of higher plants has been accomplished so far only intobacco.Prior Studies on the Chloroplast Genome. To date, stable integration ofa foreign gene into the chloroplast genome of a higher plant has beenreported only in tobacco. This was achieved with a vector which wasspecific for tobacco and which was derived from the tobacco chloroplastgenome, that is, the vector contained a sequence homologous only to thetobacco chloroplast genome and which is not highly conserved in thechloroplast genomes of other plants. Such vector is unsuitable forstably transforming plant species other than tobacco. The only publishedreport of foreign gene expression in a plant species other than tobaccois that of wheat leaves and embryos (Daniell et al., 1991), but stableintegration was not accomplished. Stable integration of a foreign geneinto the chloroplast genome of a monocotyledonous plant has never beenreported. At least in cereals (monocots), previously developedtransformation/regeneration protocols may not be amenable to plastidtransformation due to inherent inefficiencies within those systems.Also, sequential/serial selections (repeated selections), deemedimportant for achieving homoplasmy (Daniell, 1997), may not be feasibleusing those regeneration systems employed. Recent development of uniquecorn (Rudraswamy, 1997) and rice (unpublished)transformation/regeneration protocols have the potential to exhibitsubstantially increased efficiencies and allow more than one round ofselection during regeneration.

Maliga et al. in U.S. Pat. No. 5,451,513 and Svab et al., 1990 propose atransformation of the plastid genome of tobacco by a non-lethalselection technique which employs plastid DNA encoding a non-lethalselectable phenotype. According to Maliga et al. a non-lethal selectionis absolutely essential for obtaining transplastgenic lines.

Unlike the Maliga et al. technique, the method of the invention providesa selection which is lethal to all non-transformed plants, but fortobacco. Only the transformed plants survive and continue to grow. Thislethal selection takes place with virtually all antibiotics, includingspectinomycin and streptomycin in a medium containing the antibiotic ina concentration of 500-1,000 μg/ml. Similar conditions were shown to benon-lethal for tobacco by Maliga et al. Moreover, unlike the techniqueof Maliga et al., in accordance with the invention, transformation tohomoplasmy can be achieved even in the first round of selection.

In European Patent Application No. 0 251 654, Cannon et al. describetransposon-mediated chloroplast transformation of tobacco for instance,using the bacterial transposon Tn5. The vector containing the transposonis targeted at a chromosomal region known to be a “transcriptionallysilent” region in order to preserve the transcriptional integrity of thenative genes. Such a transcriptionally silent region is identified to belocated between two known divergent promoters of chloroplast genes, e.g.the promoters for the genes for the chloroplast large subunit ofribulose bisphosphate carboxylate (RbcL, “LS RuBis Co”) and for β-ATPase(atpB). These promoters transcribe the genes in opposite directions awayfrom the silent region of the chromosome. No transcription terminator isprovided in the expression vector of Cannon et al., such terminatorregions are known to be absolutely essential for gene expression inplastids. Finally, no stable chloroplast transformation is shown to beaccomplished by Cannon et al.

The invention described herein has several distinguishing features overCannon et al. The invention teaches stable transformation transmittableto the progeny. The integration is not directed into a transcriptionallyinactive region of the chloroplast chromosome. The invention integratesa cassette (which contains a transcription terminator as describedfurther hereinafter) into a transcriptionally active region of thechloroplast genome. Promoters controls the expression of one or moregenes. Unlike Cannon et al. no transposon is involved in thetransformation of the chloroplast in accordance with the invention.

In NATO Asi Series, Daniell et al., 1994 report engineering insectresistance via chloroplast genomes showing the expression of CryII-Aprotein in plants to control insects. McBride et al, 1995, and U.S. Pat.No. 5,545,818 (1996), confirm report of Daniell et al and show theexpression of Bacillus thuringiensis CRYIAc protein into plant plastids.The vectors reported by McBride are designed to introduce the constructonly into the tobacco chloroplast genome.

The Need for a Vector to Transform a Variety of Plants. It is evidentfrom the state of the art that an important need exists for achloroplast integration and expression vector for transforming,preferably stably, the chloroplast genomes of many different species ofplants. Such a “universal vector” would permit the transformation of thechloroplast genome of a selected target plant with a heterologous(foreign) DNA coding sequence and eliminate the need to constructvectors which each one is specifically suited to transform thechloroplast genome of the particular plant species which is to betransformed.

The problem to construct such a universal vector competent to transformdifferent plants has to the knowledge of the inventor, not yet beensolved.

Prior Art Concepts of the Intergenic Spacer Region. While the nucleotidesequence of coding regions of the genome, including the chloroplastgenome, are often conserved between species, in contrast the sequencesflanking functional genes, i.e. the spacer regions between codingregions typically are not conserved. The accepted dogma for lack ofconservation, and thus the low degree of homology between species ofspacer regions, is that the spacer regions typically do not performessential functions. Therefore, there is little, if any, selectivepressure to conserve the sequence of spacer regions between species. Thesequence of the spacer regions may be altered without undesirableeffects.

Stummann et al., 1988, disclose that the gene order of the ribosomal RNAoperon of the chloroplast genome is the same between different speciesof plants, including tobacco, maize, and a liverwort, Marchantia, andthat the coding sequences of this operon are highly homologous. Stummannalso discloses that the interspecies homology of the operon is less thanthe interspecies homology of the gene coding regions. This is consistentwith the lack of conservation of spacer regions; and suggests that theinterspecies homology of spacer regions in the ribosomal RNA operon isrelatively low.

The invention, contrary to the dogma of lack of conservation of thespacer regions, uses spacer regions that are highly conserved betweendifferent plants to construct vectors competent to transform a varietyof plants.

OVERVIEW OF THE INVENTION

The invention provides universal chloroplast integration and expressionvectors which are competent to stably transform and integrate genes ofinterest into chloroplast genome of multiple species of plants.Transformed plants and their progeny are provided. Monocotyledonous anddicotyledonous plant are transformed which have never been transformedheretofore. Plants transformed with a synthetic gene express valuablebiodegradable protein-based polymers (PBPs). Transformed plants producehigh value molecules. Resistance is provided to agricultural cropsagainst the major classes of chemical herbicides. Herbicide resistanceis used as a lethal selectable marker for chloroplast transformation.The transformed plants are capable of expressing in addition to thetargeted trait, a desirable, secondary non-targeted trait. Insectresistance is provided to transformed plants, both against insects thatare susceptible to Bt toxins and against insects that have developed aresistance to Bt toxins.

SUMMARY OF THE INVENTION

The Intergenic Spacer Region. The Invention's Concept.

It has been discovered contrary to the conventional belief, that thechloroplast (ct) genome of plants contains spacer regions with highlyconserved nucleotide sequences. The highly conserved nature of thenucleotide sequences of these spacer regions of the chloroplast genomemakes such spacer regions, it has been discovered, ideal for theconstruction of vectors to transform chloroplasts of widely varyingspecies of plant, this without the necessity of constructing individualvectors for different plants or individual crop species, which wouldrequire first a determination of the DNA sequence of each of thechloroplast genomes. This finding has numerous useful consequences andimportant practical applications.

The Several Embodiments of the Invention

The Universal Vector. The invention has several useful embodiments. Theinvention provides a universal integration and expression vectorhereinafter referred to as “UV” and its use for the expression of atleast one phenotype in a variety of different plants.

The integration expression universal vector of the invention comprisesan expression cassette (further described below) which comprises thenecessary genetic elements to transiently or preferably stably transformthe plastids e.g. chloroplast genome of a target plant cell with aforeign (heterologous) DNA coding for a molecule of interest, like aphenotype to be expressed by the plant or a non-plant high valuemolecule, like a biologically active peptide (or polypeptide). Theuniversal vector is constructed with a transcriptionally active regionof a chloroplast genome that is highly conserved in a broad range ofchloroplast genomes of higher plants. Preferably that region is thespacer 2 region; the intergenic spacer region between the t-RNA^(Ile)and the tRNA^(Ala) region. Such region is often referred to herein as a“spacer” region because in the chloroplast genome it is intergenicbetween several genes in the rRNA operon which is transcribed by onepromoter. When built into the universal vector such region is generallyreferred to herein as a “border” or preferably as a “flanking sequence”or “flanking sequences”. This is because in the universal vector, theoperably joined genetic elements for transforming stably the plastid ofthe target plant are flanked on each side by a sequence i.e. a fragmentof the spacer region. The flanking sequences in the vector and thespacer sequences in the chloroplast genome have sufficient homology toeach other to undergo homologous recombination. The universal vector isinserted into the spacer of a transcriptionally active region in thechloroplast genome. Generally, the spacer region is positioned in theinverted repeat region of the chloroplast genome. The rest of theconstruct, i.e. other than the flanking sequences and the expressioncassette, is generally referred to herein as the “vector” whichcomprises bacterial sequences, like the plasmid cloning vectors pUC,pBR322, pGEM or pBlueScript.

The Expression Vector or Cassette. The universal vector comprises anexpression cassette which is flanked on each side by a flankingsequence. A suitable expression cassette for use in the invention isdescribed in U.S. Pat. No. 5,693,507 (1997), which is incorporatedherein by reference. That cassette comprises, operably joined, atranscriptional initiation region functional in plant chloroplast, atleast one heterologous DNA sequence coding for a target molecule ofinterest, e.g. a gene (or functional fraction thereof) encoding abiologically active compound, and control sequences positioned upstreamfor the 5′ and downstream from the 3′ ends and a transcriptiontermination region to provide expression of the coding sequence in thechloroplast genome of a target plant. Preferably, the expressioncassette is flanked by plant DNA sequences, like chloroplast DNAsequences, in order to facilitate stable integration of the expressionvector into the chloroplast genome. In the construction of theexpression cassette, the DNA sequence comprises one or more cloningsite(s) for integration of the gene(s) of interest.

The spacer sequences that have been identified in plastids of higherplants are ubiquitously conserved between a great variety of plants.These sequences were found to be ideal to construct the universalvectors of the invention which are, as a result, competent to transformthe chloroplast genome of a large variety (or multiplicity) of targetplants by homologous recombination. It is thus immaterial from whichindividual spacer of a particular plant the universal vector isconstructed.

As is known, it will be generally advisable to have at least oneadditional heterologous nucleotide sequence coding for a selectablephenotype, such as a gene providing for antibiotic resistance or afunctional portion thereof to serve as a marker associated with theexpression cassette or with the universal integration expression vector.This facilitates identification of the plant cells in which the foreigngene has been stably integrated. Marker genes are known in theliterature, for instance β-lactanase, herbicide resistant genes such asthe mutant psbA gene or EPSPS-aroA, the cat gene which encodeschloramphenicol acetotranferase, and the uidA gene encodesβ-glucuronidase (gus) and others.

It is recognized that tobacco is unique in being not susceptible to thelethal affect of streptomycin and spectinomycin. Though tobacco leaveslack the pigmentation when exposed to a medium with such an antibiotic,continued growth is observable. However, this property of tobacco isreadily circumvented. There are numerous antibiotics available which arelethal for tobacco, like hygromycin. Another approach is to select agene which expresses a visible marker like a color, fluorescence, etc.,like the reporter gene mGFP, that codes for a green fluorescent protein.

Method of Transformation. The invention provides a transformation methodwhich can produce homoplasmy (integration of foreign genes into all ofthe chloroplast genomes of the plant cell) after a first round ofselection without the need for a further selection process. The methodfor transforming a plant uses the universal vector constructed withflanking sequences from a plant species other than the species of thetarget plant to be transformed. Alternatively, the vector may containflanking sequences from the same plant species as the target plant,including from tobacco.Method to Construct the Universal Vector. The invention further providesa method to construct the universal chloroplast integration andexpression vector. To this effect, a spacer portion of the chloroplastgenome from any plant is determined to be highly homologous to more thanone species of plants. A nucleotide sequence corresponding to thatspacer region is obtained from the identified chloroplast genome (orsynthesized) and is incorporated into a suitable vector, such as bysubcloning into a plasmid. The spacer region is positioned as flankingsequences to the expression cassette comprising the necessary geneticelements for transformation of the plastid and expression of the foreigngene(s).

Any method of transformation of the chloroplast may be used. Any gene(or functional portion thereof) which may be utilized to transform aplant chloroplast and encode a desired peptide to confer the desiredtrait to the target plant is suitable for transformation with theuniversal vector.

Transformed Plants. The invention further provides plants in which thechloroplast genome has been stably, that is, permanently transformedwith the universal vector of the invention, including the progenythereof.

The invention includes monocotyledonous plants like cereals or plantcells, such as maize, rice, barley, oat, wheat and grasses, and theirprogeny in which the chloroplast genome has been stably transformed withthe universal vector derived from the same species or from a differentspecies than the transformed plant. The invention providesdicotyledonous and monocotyledonous plants, stably transformed followinga single round of selection, due to homoplasmy achievable with theuniversal vector comprising a chloroplast origin of replication (ori).The invention also provides stably transformed plants of differingspecies, including varieties of the same species, genera, families,orders, and divisions of plants.

In accordance with the invention, a plant in which the chloroplastgenome has been stably transformed with one or more foreign genes ofinterest includes mature plants and progeny thereof, like seedlings andembryos. The term “plant” in this context also includes portions ofplants such as explants like cuttings, tissue cultures, cellsuspensions, and calli.

Thus, the invention includes the stably transformed multicellularplants, their progeny, the seed, and the transformed plastids, e.g. thechloroplast, etc., and method of regenerating the transformed plants.

In this specification and in the claims, when reference is made todifferent “species”, the term “species” refers not only to “species” butto varieties within a species, genera, families, order, and divisions ofthe plant kingdom. Thus, a universal vector which can be used totransform plants of different species is understood to be able totransform plants of different varieties within a species, differentgenera, different families, different orders, and different divisions.The term “plant” (or “plants”) is intended to be generic as used herein.

Expression of Non-plant Products

Biopolymer genes. Another embodiment of the invention using theuniversal integration and expression vector provides plants transformedwith a synthetic biopolymer gene that codes for biodegradableprotein-based polymers (PBPs).

These polymers have important properties of practical importance,discussed hereinafter.

Production of High Value Molecules-Biologically Active Molecules. Theintriguing discovery that transformation with a synthetic gene whichneed not have a natural analogue in plant or animal, to produce PBPs, isfeasible, has shown the wide applicability of the vector in yet anotherfield of human endeavor: the production of biologically activemolecules, like pharmaceuticals in plants, from any gene or functionalfraction thereof, synthetic or natural.

A further embodiment of the invention is therefore the use oftransformed plants as bioreactors (as factories) for biopharmaceuticals.There are at least two capabilities often needed for the production ofproteins of pharmaceutical value, not possible in prokaryotic systems.Plants, unlike bacteria, are able to produce the foreign protein in abiological active conformation. Plants are also often more tolerant tothe alteration of their biosynthetic pathways. Thus, the plants can betransformed with a gene non-functional in (or foreign to) plants, thatmay be synthetic or not, that may normally be functional (competent) inanimals (mammals), in oviparous, in pesces or other species.

The invention further provides transformed plants comprising a geneprovided by an expression cassette, preferably by a universal vector,which codes for a variety of desired products, especially biologicallyactive molecules like peptides (polypeptides), proteins, insulin, humanserum albumin (HSA) and other molecules further described hereinafter.The plants are allowed or caused to grow, and the products are isolatedfrom the transformed crop, like tobacco, maize, etc., and if desirable,harvested first and if necessary, purified.

Herbicide Tolerance. Another important embodiment of the inventionprovides transgenic herbicide resistant plants in which a foreigntransgene conferring resistance to one or more herbicides areintegrated, preferably stably, into the chloroplast genome by means ofthe universal vector. Of particular importance are transformed plantswhich exhibit glyphosate resistance and thus be resistant to “ROUNDUP™”,a herbicide available commercially from Monsanto Company. The universalvector provides an effective means to transform the chloroplast genomeof any plant and to confer resistance (or tolerance) to any of theherbicidal chemicals.

A different aspect of the invention provides a method to transform aplant by means of an expression cassette, preferably by means of theuniversal vector, to cause it to produce a non-targeted (secondary orother) trait (or phenotype). [See, for example, Penazloza, V., et al.(1995), who report that expression by gromycin β-phosphotransferace geneconfers resistance to the herbicide glyphosate.]

In another aspect of the invention, herbicide tolerance is used as amarker gene for chloroplast transformation.

Insect Resistance. A further embodiment of the invention provides insectresistance. With the increased concerns of using chemical pesticides,the use of Bacillus thuringiensis (Bt) formulations has been widelyadvocated. Bacillus thuringiensis produces many types of crystallineinclusions which are toxic to insects. The proteins comprising theseinclusions have been categorized based on insecticidal host range, andprotein homology. The CRYI and CRYII toxins have insecticidal activityagainst lepidoptera, or lepidoptera and diptera, respectively. CRYIprotoxins are 130-135 kDa in size which are enzymatically cleaved intoproteins of 65 kDa for insecticidal activity. CRYII protoxin is 65 kDain size with a protein with a molecular mass of 60-62 kDa forinsecticidal activity. Many commercially important insects pests(especially in the family Pyralidae) are susceptible to CryIIA toxin,including European corn borer, Ostrinia nubilalis, lesser cornstalkborer, Elasmopalpus lignosellus, cowpea pod borer, Maruca testulalis,tobacco budworm, Heliothis virescens, tobacco hornworm, Manduca sextaand gypsy moth Lymantria dispar, Daniell et al. 1994.

However, Bt formulations have not been as effective as anticipatedprimarily due to their susceptibility to UV radiation, inadequatecoverage, expense and limited host range. Delivery of Bt toxins viaBt-transgenic plants is therefore appealing.

Acceptable insect control has occurred with nuclear-transgenic Bt cottonagainst the tobacco budworm, Heliothis virescens, but these plants donot express enough Bt toxin to control the cotton bollworm, Helicoverpazea. Additionally, these Bt genes may outcross to related plant speciesvia pollen. To circumvent these concerns, chloroplast transformation andexpression has been evaluated in accordance with the invention becauseof the following reasons. 1) Plant cells containing chloroplasts cancontain up to 10,000 gene copies per cell, 2) chloroplasts can readintact bacterial DNA, including operons, and 3) chloroplast genomes arematernally inherited, and therefore, foreign gene escape via the pollenis drastically eliminated because chloroplast DNA is generally degradedin pollen.

CRY2A was chosen because CRY2A is relatively non-homologous to CRY1A andtherefore, displays only slight cross resistance against someCRY1A-resistant H. virescens populations. Because CRY2A is a “naturallytruncated” high expression levels may be achieved without sacrificingthe 3′ region.

According resistance has been provided to tobacco chloroplast genometransformed with a universal vector of the invention to insects normallysusceptible to Bt toxins and also to insects that have developedresistance or less susceptibility to Bt toxins. Insects that had neverbeen killed by any CRY toxin showed 100% mortality on transformedtobacco.

The expression of cry2A in a plant could therefore be a valuable tool incontrolling multiple insect pests while overcoming the development of Btresistance. Other insecticidal proteins such as cholesterol oxidase thatkill boll weevil by a different mechanism, are expressed to high levelsin chloroplasts.

Other embodiments of the invention will become apparent hereinafter.

DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a map of the tobacco chloroplast genome. The thick lines inthe genome map represent the inverted repeat regions of the chloroplastgenome. The arrows labeled “UV” represent the insertion sequences forthe preferred embodiment of the universal integration and expressionvector (UV); the arrow labelled “TV” represents the insertion sequencefor the tobacco vector (TV).

FIG. 2A shows the tobacco chloroplast vector (TV) pZS-RD-EPSPS forexpression of herbicide resistance.

FIG. 2B shows the universal chloroplast expression and integrationvector (UV), pSBL-RD-EPSPS for expression of herbicide resistance.

FIG. 3A shows the universal chloroplast integration and expressionvector, pSBL-CG-EG121 for biopolymer expression.

FIG. 3B shows the tobacco integration and expression and vector,pZS-CG-EG121 for biopolymer expression.

FIGS. 4A-4E show the sequence homology (SEQ ID NO: 1, SEQ ID NO: 2, andSEQ ID NO:3) of spacer regions between tobacco and other crop species. Asite for foreign gene insertion is shown by arrows. Upstream of the sitefor foreign gene insertion the site of an origin or replication (ori) isshown.

FIGS. 4-F-4G show the sequence alignment SEQ ID NO: 4 (Epifagus), SEQ IDNO: 5 (Tobacco), SEQ ID NO: 6 (Helianthus), SEQ ID NO: 7 (Oenothera),SEQ ID NO: 8 (Alnus), SEQ ID NO: 9 (Rice), SEQ ID NO: 10 (Maize), SEQ IDNO: 11 (Soybean), SEQ ID NO: 12 (Pea), SEQ ID NO: 13 (Spinach), SEQ IDNO: 14, SEQ ID NO: 15 (Tobacco) and SEQ ID NO: 16 (Cuscuta) of thespacer (64 bp) region 16S-23S rDNA from several crop species with thetobacco chloroplast sequence where (+) represents the positive and (−)the negative strands, respectively.

FIG. 5 shows construction of the vector pSBL-Ct border.

FIGS. 6A-C shows construction of the vector pSBL-CtV1 selectable markergene cassette containing a chloroplast 16S rRNA promoter (Prrn), theaadA gene and a 3′ untranslated region of the chloroplast psbA gene.

FIGS. 7A-D shows vectors pSBL-CtV2, pSBL-CtV3, pSBL-CtVH, pSBL-CtVHF,respectively.

FIGS. 8A and 8B show vectors pSBL-CtVHBt and pSBL-CtVHBtR, respectively.

FIG. 9 shows transformed and untransformed tobacco plants growing in thepresence of spectinomycin indicating non-lethal selection on the medium(500 μg/ml). Note growth of transformed (bleached) and untransformedleaves (green).

FIGS. 10A-G show corn plastid transformation and regeneration.

FIG. 11 shows corn plastid transformation. Transformed corn plants grownormally (middle shoot) while untransformed plants die on the lethalmedium, confirming lethal selection by the antibiotic, (1000 μg/mlspectinomycin).

FIGS. 12A-F shows rice plastid transformation and regeneration.

FIGS. 13A and 13B shows PCR analysis of DNA isolated from the leaves ofrice transformants.

FIG. 14 shows peanut plastid transformation. Transformed peanut plantsgrow normally (middle and on left side of plate) while untransformedplants die in the lethal medium (500 μg/ml spectinomycin).

FIG. 15 shows soybean plastid transformation. Two transformed plantsshow shoots, the other plants die on the lethal medium, confirminglethal selection by the antibiotic (500 μg/ml spectinomycin).

FIG. 16 shows sweet potato embryo transformation on lethal selectionmedium (500 μg/ml spectinomycin).

FIG. 17 shows grape cells transformation. The transformed culture cellsbecome green while the untransformed cells die in the lethal selectionmedium (500 μg/ml spectinomycin).

FIG. 18 shows protein-based biopolymer (PBP) expression by chloroplastintegration and expression vectors in E. coli.

FIGS. 19A-B show Southern blot analysis performed with the transformantsfrom PBP transgenic plants using the tobacco vector (TV). Probes werefrom chloroplast border sequences (A) or from polymer (EG121) genesequences (B).

FIGS. 20A-B show Southern blot analysis performed with the transformantsfrom PBP transgenic plants using the universal vector (UV). Probes werefrom chloroplast border sequences (A) or from the aadA gene (B).

FIG. 21A shows foreign gene transcript levels analyzed by northernblotting using total RNA isolated from the control, chloroplasttransformants and a nuclear tobacco transgenic plant highly expressingthe synthetic biopolymer gene (EG121).

FIG. 21B shows an enlargement of lanes 4-7 of FIG. 21A.

FIG. 22 shows Western blot analysis of purified polymer protein fromtransgenic plants.

FIG. 23A shows the higher growth rate of E. coli containing the tobaccovector with the EPSPS gene.

FIG. 23B shows the higher growth rate of E. coli containing theuniversal vector with the EPSPS gene.

FIGS. 24A-B show the integration of foreign genes into the plastidgenome by PCR using rbcl and aadA primers (A), or 16SRNA and aadAprimers (B).

FIGS. 25A-C show the integration of the aroA gene into the chloroplastby Southern analysis and the high generation of homoplasmy using EPSPSprobe (A) or rcbL-orf512 probe. The site of integration is shown in (C).

FIGS. 26A and 26B show generation of seeds collected from control andtransformed tobacco plants, respectively, in the presence of theselectable markers.

FIGS. 27A and 27B show transgenic and control tobacco plants sprayedwith glyphosate.

FIGS. 28A and 28B show tobacco susceptibility (control) and resistance(transformed) to insects.

FIG. 29 shows (Western blot analysis) total protein isolated fromcontrol and transgenic tobacco plants.

Preferred embodiments of the invention are described in greater detailhereinafter.

DETAILED DESCRIPTION OF THE INVENTION

The Universal Integration and Expression Vector. The universalintegration and expression vector of the invention is competent forstably transforming chloroplasts of various different target plants.Heterologous DNA coding sequences can be provided in the cassette tocode for a phenotype such as herbicide resistance, insect resistance orother traits. The vector further comprises a flanking sequence on eachside of the DNA coding sequence which is homologous to a spacer sequenceof the chloroplast genome, which spacer sequence is conserved in thechloroplast genomes of different plants. In this manner, stableintegration of the heterologous gene into the chloroplast genome of thetarget plant is facilitated through homologous recombination of theflanking border sequences with complimentary spacer sequences in thechloroplast genome. The universal vector is competent to transform anyplant species.

The trnI and trnA spacer region, has been found to be highly conservedin a great variety of plant species, from cyanobacteria to higherplants, such as monocotyledonous and dicotyledonous. The trnI and trnAgenes flank on each side a spacer region, referred to as spacer 2 “or‘spa 2’ region” (FIG. 4F-4G). The regions on either side of the spacerregion likewise have almost perfect homology with corresponding regionsbetween species of plants from cyanobacteria to higher plants, exceptthat the higher plants contain two introns in the trnI and trnA genes.Longer border sequences tend to favor more efficient integration offoreign DNA. Therefore, although not essential, the homology betweenspecies of the trnI and trnA genes, in addition to the homology of thespacer region, contributes to the efficiency of transformation andintegration (FIG. 4A-4E).

If longer border sequences which include non-homologous portions, areincorporated into the vector, the non-homologous portion of the sequencewill be “looped out” and “clipped off”, in the recombination process andwill not integrate into the target chloroplast genome.

Different universal vectors can be constructed with the spacer region.For instance, shorter or longer flanking sequences can be constitutedwith part or all of the trnA and trnI genes adjacent to spa ‘2’.

A preferred universal vector comprises the flanking sequences and anexpression cassette which comprises the following genetic elements toprovide for transcription and translation of the DNA coding sequenceorganized in the following order (from the 5′ to the 3′ ends): a 5′ partof the flanking sequence, a promoter functional in chloroplast, a DNAsequence with appropriate cloning site(s) for insertion of one or morecoding sequence(s) for the desired phenotype or molecule of interest,and for a selectable marker, a terminator of transcription and a 3′ partof the flanking sequence. The order of the DNA sequences coding thedesired phenotype and the selectable marker can be switched. Additionalflanking plant DNA sequences can be provided to promote stableintegration. Preferably, the flanking sequence comprises an origin ofreplication (ori).

In a particular illustration, the highly conserved spacer region residesin the inverted repeat of the chloroplast genome. However, theparticular location of the spacer in the chloroplast genome is not asimportant as its high homology with the spacer region of differentplants.

Further, as may be seen in FIGS. 4F-4G, the spacer 2 (or spa 2) sequencewhich is 64 bp long is too short to include the chloroplast genome oriwhich resides upstream, of that spacer. If it is desired to include theori, a longer spacer sequence encompassing the ori will be selectedwhich will include the spacer sequence and an additional sequence in theflanking sequences. This will provide a longer template for homologousrecombination into the recipient chloroplast genome and promotehomoplasmy.

Another preferred vector is one in which the flanking sequencescomprise, each one, in addition to the spacer 2 region, a portion or allof the intergenic spacer region between the tRNA^(Ile) and thetRNA^(Ala) genes of the chloroplast genome (FIG. 4A-4E). Further, theflanking sequences may include part or all of the tRNA^(Ile) and thetRNA^(Ala) genes, respectively. Optionally, the flanking sequencescomprise each one part or all of the 16S and/or 23S rRNA gene sequences.

Illustrative Universal Vectors. A preferred universal vector comprises aDNA sequence which comprises the sequence of the spacer 2 region betweenthe highly conserved trnI and the trnA genes between the 16S-23S rRNAgenes of the chloroplast genome. Preferably, this region comprises partor all of the DNA sequence (FIG. 4F-4G) of the trnI and the trnA genes.That region is excised from a selected plant like tobacco and subclonedinto a commonly available plasmid like pUC19, e.g. at the PvuII site.Into the plasmid there is inserted the expression cassette whichcontains a selectable marker gene, a chloroplast 16SrRNA promoter, agene encoding an enzyme conferring a resistance to an antibiotic likethe aadA gene encoding aminoglycoside 3′ adenyl transferase conferringresistance to streptomycin/spectinomycin and 3′ untranslated region ofthe chloroplast psbA gene.

Specifically, when a universal vector is constructed with plasmidpSBL-Ct-bor (FIG. 5), a selectable marker gene cassette containing achloroplast 16S rRNA promoter, the aadA gene encoding aminoglycoside3′-adenyl transferase conferring resistance forstreptomycin/spectinomycin, or a herbicide resistance gene and a 3′untranslated region of the chloroplast psbA gene, was inserted into theplasmid. (FIG. 6) This selectable marker gene cassette was then insertedinto the universal border in two different orientations. In the vectorpSBL-CtV1, the selectable marker gene cassette was inserted into thetrnI gene (FIG. 6A). In the vector pSBL-CtV2 (FIG. 7A), the selectablemarker gene cassette was inserted between the trnI and trnA genes in thespacer region, in the direction of the 16S rDNA transcription. In thevector pSBL-CtV2 R (map not shown), the selectable marker gene cassettewas inserted between the trnI and trnA genes in the spacer region, inthe direction opposite of the 16S rDNA transcription.

Several genes of interest have been inserted into the pSBL-CtV2 vector,a preferred embodiment of the universal vector. For example, the vectorpSBL-CtV3 (FIG. 7B) contains the reporter gene mGFP that codes for agreen fluorescent protein, isolated from jelly fish. This gene may alsobe useful for visible selection of transformed plants or in ornamentalhorticulture, for example, in ornamental crops like Christmas trees oreven lawn grass, which may glow with green fluorescence uponillumination with blue light.

The vector pSBL-CtVH (FIG. 7C) contains a different selectable marker,hygromycin phosphotransferase (hph gene driven by the chloroplast atpBgene promoter), which confers resistance to the antibiotic hygromycin.This vector may be used to transform plants that are resistant to otherantibiotics and is particularly useful for transforming monocots, whichare generally resistant to other commonly used antibiotics. This genemay confer additional traits such as herbicide resistance, anon-targeted trait.

Vector pSBL-CtVHF (FIG. 7) contains the GFP and hph genes, which can beused for lethal or a combination of lethal/visible selection.

A Chloroplast Vector Specific for Tobacco and a Universal ChloroplastVector. The tobacco chloroplast vector pZS-RD-EPSPS (FIG. 2A)(“TV”) andthe universal vector pSBL-RD-EPSPS (FIG. 2B)(“UV”) contain both the Prrnpromoter (of the 16S rRNA), the aadA gene (for spectinomycin selection),the mutant aroA gene that codes for the enzyme EPSPS synthase (forglyphosate selection) and the psbA 3′ region. The flanking sequences inpZS-RD-EPSPS contain rbcL and orf 512 and in pSBL-RD-EPSPS contain thetrnI and trnA genes facilitating integration into either the LargeSingle Copy region (FIG. 1 at the “TV” arrow) or the inverted repeatregions (FIG. 1 at the UV arrows) of the tobacco chloroplast genome,respectively.

Glyphosate is the active ingredient in Monsanto's herbicide ROUNDUP™ andis used as a selectable marker for herbicide selection of transgenicplants.

Construction of Chloroplast Vectors. Standard protocols for vectorconstruction including Klenow filing the dephospphorylation, were used.The tobacco chloroplast expression vector pZS-RD-EPSPS is shown in FIG.2A. The universal chloroplast vector PSBL-RD-EPSPS is shown in FIG. 2A.The construction of these vectors is further shown in the examples. Bothplasmids were amplified in the XL1 Blue Strain of E. coli. Growth curveswere recorded in M-9 minimal medium. Both vectors are used for selectionon glyphosphate to confirm resistance to ROUNDUP™.

The chloroplast expression vector pZS-RD-EPSPS is specific for tobaccoand as noted earlier is not useful to transform other plants (Maier etal., 1995). In contrast, the universal chloroplast expression andintegration vector PSBL-RD-EPSPS (FIG. 2B) is competent to transformchloroplast genomes of numerous other plant species because of theuniversality of the vector as described above. The universal vectorintegrates foreign genes into the 16S-23S spacer region of thechloroplast genome. The universal vector uses the trnA and trnI genes(chloroplast transfer RNAs) coding for alanine and isoluceine) from theinverted repeat region of the chloroplast genome as flanking sequencesfor homologous recombination. The chloroplast border sequence used inthis invention also contains the chloroplast origin of replication(oriA), as confirmed in several crop species including pea (Nielsen etal., 1993) and tobacco (Lu et al., 1996), which may explain the highlyconserved sequence homology in this region. This origin of replicationprovides increased number of plasmid templates for efficient integrationinto the recipient chloroplast genome and achieve homoplasmy.

As shown above, in the construction of the universal vector, anexpression cassette containing a chloroplast promoter, a selectablemaker gene conferring resistance to an antibiotic (or other selectedmarker), a gene encoding the target molecule, and the other elements (asdescribed herein) are inserted at a convenient restriction site into theDNA fragment containing the spacer region. If desired, the foreign geneencoding the target molecule may be inserted into the expressioncassette after insertion of the cassette into the DNA fragmentcontaining the conserved spacer region so that, before insertion, thecassette will include multiple cloning sites for insertion of one ormore DNA coding sequences.

The position of the restriction site in the spacer sequence candetermine the respective length of the two flanking sequences, whichwill be fractions (of different or same length) of the spacer region.Thus, the two flanking sequences need not be identical in length as longas each one contains enough of complementarity to the target chloroplastgenome to promote homologous recombination.

Because the vector of the invention has such a high degree of homologyto the spacer region of the chloroplast genomes of multiple species ofplants, it is competent to transform, not only the species of plantsfrom which the border sequence of the vector is derived, but any plantspecies.

As used in this specification, the term “homologous” means that a DNAsequence from one plant species possesses regions of sequence identityto portions of a DNA sequence from another plant species. That is, iftwo DNA sequences are highly homologous, the DNA species may have 100%sequence identity or less than 100% identity. For example, for purposesof chloroplast genome integration, a 400 bp sequence which is only 25%homologous overall, but which contains a 100 bp portion which is 85% to90% or more homologous, is considered to be highly homologous with thechloroplast genome.

The inclusion of a chloroplast ori within the flanking sequences of theuniversal vector has also been shown to be beneficial. Without beingbound by theory, it is believed that the presence of the ori in theuniversal vector promotes homoplasmy following a single round ofselection, without the need for a second selection step. This isespecially important in the transformation of monocotyledonous plants,such as maize or rice, in which a second selection step is not feasibledue to the difficulty of growing these plants in culture from leafcuttings with the resultant need to grow these plants from embryos. Ifan ori is desired but is lacking, it may be introduced into a flankingsequence or elsewhere. If it is desired to increase the copy number ofthe introduced universal vector, a chloroplast DNA fragment containingan ori will be inserted outside the flanking sequences so that it willfunction only to amplify the copy number of the universal vector anddoes not become integrated into the chloroplast genome.

As opposed to being derived from a specific plant, the flankingsequences can be derived from a spacer region synthetically made asshown below.

For transcription and translation of the DNA sequence encoding thepolypeptide of interest, the entire promoter region from a gene capableof expression in the chloroplast generally is used. The promoter regionmay include promoters obtainable from chloroplast genes, such as thepsbA gene from spinach or pea, the rbcL and atpB promoter region frommaize and rRNA promoters. Competent promoters are also described andother literature sources are identified in U.S. Pat. No. 5,693,507.

The flanking sequences shown by U.S. Pat. No. 5,693,507 and the otherpublications to promote stable integration are not the flankingsequences of the universal expression and integration vector describedherein which are highly conserved from plant species to plant species,whereas the flanking sequences of that patent and the other publicationsare not.

Identification of Intergenic Spacer Sequences. The invention providesmethods to identify appropriate untranscribed intergenic spacersequences in plants which are appropriate to construct the universalvectors. The method comprises isolating plastid genomic DNA, carryingout hybridization with a radioactive labeled probe of a known spacer,detecting and isolating plastid sequences which exhibit the desireddegree of homology with the probe. As an illustration, to determine if aplastid genome of unknown structure and sequence possesses the spacerregion, Southern blots utilizing the tobacco spacer region as a probeare carried out. Plastid genomic DNA is isolated and cleaved by anappropriate restriction enzyme according to established procedures.Hybridization with the spacer probe is conducted under both stringent(e.g., 50% formamide at 68° C., wash in 0.1×SSC at 68° C.) andnon-stringent condition (e.g., 6×SSC, at 68° C., wash in 2×SSC at 50°C.)(1×SSC is 0.15M NaCl, 0.015M sodium citrate) to detect plastidsequences exhibiting approximately 90-100% homology or 60-100% to thetobacco spacer, respectively. The identified plastid sequences are thenisolated. If one's requirement of homologous recombination is morepermissive, a lower degree of hybridization to the probe, such about 60%can be satisfactory.

Thus, any known or unknown spacer region of sufficient homology forrecombination is suitable for the construction of the UV. Likewise, theknown sequence of any intergenic highly conserved spacer sequence may beused to identify and isolate plastid sequences which are homologous to aknown spacer sequence.

Alternately, the BLAST program as described herein-above can be used toidentify highly conserved regions in plastid genomes of which thesequences are known.

Plants that can be Transformed. Plants which may be transformed by theuniversal vector of the invention include any lower plants, such ascyanobacteria, any higher plants, such as monocotyledonous anddicotyledonous plant species. The plants to be transformed can besolonacious plants or plants that grow underground. A non-exclusive listof examples of higher plants which may be transformed with the universalvector includes cereals such as barley, corn, oat, rice, and wheat,melons such as cucumber, muskmelon, and watermelon; legumes such asbean, cowpea, pea, peanut; oil crops such as canola and soybean;solanaceous plants such as tobacco, tuber crops such as potato and sweetpotato, and vegetables like tomato, pepper and radish; fruits such aspear, grape, peach, plum, banana, apple, and strawberry; fiber cropslike the Gossypium genus such as cotton, flax and hemp; and other plantssuch as beet, cotton, coffee, radish, commercial flowering plants, suchas carnation and roses; grasses, such as sugar cane or turfgrass;evergreen trees such as fir, spruce, and pine, and deciduous trees, suchas maple and oak. Of greatest present interest are the majoreconomically important crops like maize, rice, soybean, wheat andcotton. None of these plants to the inventor's knowledge, other thantobacco, has ever been stably transformed via the chloroplast genome,and none, including tobacco, have been stably transformed by a universalvector, as described herein. A plant from which the DNA sequence isfrequently obtained is tobacco because it is the most thoroughlycharacterized plant, but any other plant chloroplast genome is suitable.

It will be recalled as described above that the vector used to stablytransform tobacco was not competent to transform the chloroplast genomeof other plants.

Method for Transformation. The expression cassettes may be transformedinto a plant cell of interest by any of a number of known methods. Thesemethods include, for example, the following. Transformation by tungstenparticle bombardment, polyethylene-glycol-mediated transformation, useof a laser beam, electroporation, microinjection or any other methodcapable of introducing DNA into a chloroplast. See, for example,Sanford, 1988; Daniell, 1993; Daniell, 1997; U.S. Pat. No. 5,693,507;Kin Ying et al., 1996. The use of these techniques permits theapplication of the invention described herein to a wide variety of bothmonocotyledonous and dicotyledonous plants.

Expression of Non-Plant Molecules from Transformed Plants

The increased usefulness of the universal expression integration vectorof the invention is clearly shown by the competency of the vector togenerate transformed plants to express non-plant and valuable molecules.

Biodegradable Protein-Based Polymers. In accordance with anotherembodiment of the invention, the universal vector has been used totransform tobacco with a synthetic gene expressing protein-basedpolymers (PBPs). Such polymers, and the genes expressing them, are knownin the literature. (Daniell, et al. 1997). Of particular interest, areprotein-based polymers (PBP) which have repeating pentamer sequenceslike GVGVP (Yeh et al., 1987). These PBP polymers show usefulinverse-phase temperature transition property. The protein becomeinsoluble when the temperature is raised above the transition state.PBPs offer a wide range of materials similar to that of thepetroleum-based polymers, such as hydrogels, elastomers, and plastics.They also show remarkable biocompatibility, thereby enabling a wholerange of medical applications including the prevention of post-surgicaladhesions, tissue reconstruction and programmed drug delivery (Urry etal., 1993). On the non-medical side, potential applications include usein transducers, molecular machines, superabsorbents and biodegradableplastics (Daniell et al., 1997). Such polymers include the polymer(Val'-Pro²-Glv³-Va1 ⁴-Pro)_(n) (VPGVP)_(n) SEQ ID NO: 18 wherein “n” canvary from 1 to units in the hundreds, like 250 or their analogs.Important commercial possibility and related aspects are discussed byDaniell, 1995. Useful biodegradable plastics are made from PBPs. Thegenes experiencing these PBPs are also useful in the invention in thatthey can be used as carries, i.e. the gene of a molecule of interest canbe fused to the PBP gene of chloroplast integration and expression.

In a prior study, the synthetic polymer gene coding for (GVGVP)₁₂₁ SEQID NO: 17 was hyperexpressed in E. coli to the extent that polymerinclusion bodies occupied nearly 80-90% of the cell volume (Guda et al.1951; Daniell et al. 1971). The same gene was also expressed in thenuclear compartment of cultured tobacco cells (Zhang et al., 1995) andleaves of transgenic tobacco plants (Zhang et al.), 1996)

In a model system, the intergenic region of the trnI and the trnA genesin the 16S-23S rRNA spacer region of the tobacco genome (FIG. 1) wasused to construct an universal vector for integration of the selectablemarker gene aadA gene and the synthetic polymer gene (EG121). The vectorwas inserted into the inverted repeat region of the chloroplast genome.Transformed tobacco plants expressed high level of the polymer protein.Chloroplast genome of other plant species, are also transformable withthe synthetic gene to express the protein-based polymer, using theuniversal vector.

Production of High Value Molecules. The studies with the biopolymer haveshown that a non-plant product can be expressed by a synthetic gene,thus making it possible by means of the vectors of the invention toexpress biologically valuable molecules from transformed plants with agreat variety of DNA coding sequences. The DNA coding sequence will becomprised in a universal vector, or, if desired, in an expressioncassette, as described above.

Transgenic plants are known to produce valuable biologically activemolecules by nuclear transformation but not via chloroplasttransformation. See the following literature references, all of whichare incorporated by reference. Daniell, 1995; Miele, 1997; Lyons, 1996;Daniell, and Guda, 1997; Arntzen, 1997.

Expression of Biologically Active Molecules. Plants transformed inaccordance with the invention with the universal vector or with theexpression cassette can be made to express valuable biologically activemolecules in chloroplast containing parts of the plants. The plants willthen be harvested by known practices. The transformed plants containingthese products can thus be administered orally. Arntzen, 1997.Production of pharmaceuticals by transgenic plants has been performedwith peptides (proteins) for many pharmaceutical applications, includingvaccines, immunomodulators, growth factors, hormones, blood proteins,inhibitors, and enzymes. Typical transgenic plant derived biologicalswhich have been reported, include vaccines against viral diseases; viralpeptide epitopes, like human immunodeficiency virus, non-viral peptideepitopes, bacterial antigenic proteins; bioactive peptides, recombinanttoxins, plantibodies (recombinant antibodies), serum proteins, and plantsecondary metabolites. All these products can be expressed inchloroplasts of transgenic plants in accordance with the invention.

Typical pharmaceutical peptides or proteins produced in transgenicplants include hepatitis B surface antigen, norwalk virus capsidprotein, foot-and-mouth disease virus, human rhinovirus 14, humanimmunodeficiency virus, S. mutans surface protein, E. coli enterotoxin,B subunit, malarial circumsporozoite epitopes, mouse ZP3 protein epitope(vaccine); mouse catalytic antibody 6D4, mouse mAB Guy's 13, mAB B1-8,anti-phytochrome Fv protein, anti-substance P (antibody); human serumalbumin (HSA), human protein C (serum protein); α-trichosanthin, ricin(cytotoxin); human epidermal growth factor (growth factor);leu-enkephalin (neuropeptide) and human acid β-glucosidase(hGC)(enzyme). Many of these molecules have been expressed in tobacco,potato tubers, etc.

Of particular interest, in accordance with the invention is theproduction of insulin and human serum albumin (HSA) with the universalintegration and expression vector or with an expression vector. The HSAhas already been produced in transgenic (nuclear) potato and tobaccoplants. Sijmons et al, 1990. The aforementioned products can be producedvia chloroplast transformation in accordance with the invention.

Insulin. The invention provides a method for expressing insulin as apolymer-fusion protein from a transformed plant. The plant may betransformed with an expression cassette or with the universalintegration and expression vector which comprises a synthetic biopolymergene, line (GVGVP) ₄₀ SEQ ID NO: 17. A suitable plant is tobacco becauseof the ease of genetic engineering and the need to find alternative usesof this controversial corp.

For expression in bacteria, the method comprises constructing a vectorfor expression in E. coli as a polymer (GVGVP)₄₀ SEQ ID NO: 17 genefusion with a pro-insulin gene, expressing the polymer-insulin fusionproteins in E. coli, (grown in a known manner), purifying therecombinant protein utilizing the temperature-transition properties ofthe protein-based polymer, and cleaving the insulin from the polymerusing well known methods.

For plant transformation, an expression cassette or the universalintegration and expression vector, which comprises the polymer genefusion with the pro-insulin gene, is introduced into a target plant,like tobacco. The polymer-insulin fusion protein is expressed from theplant, the polymer fusion protein is extracted from the chloroplast andthe cytosol compartments of the plant cells, the insulin is cleaved fromthe polymer protein with dithiothreitol (DTT) or the other knownmethods, and the insulin is collected. Alternatively, insulin polymerfusion product can be expressed in an edible crop or edible parts of thecrop.

The technique of fusing a DNA sequence coding for a molecule ofbiological activity to a synthetic gene expressing a protein-basedpolymer for expressing, in a suitable bacterial or yeast host or atransformed plant, is a highly promising method of wide applicability.

Recombinant Human Serum Albumin in Plants. In nuclear transgenic tobaccoand potato plants, recombinant human serum albumin (rHSA) that isindistinguishable from the authentic human protein has been produced(Sijmons et al., 1990). This showed the expression of a valuable proteinin transgenic plants, but also that it was possible to achieve properprocessing by fusion of HSA to a plant pro-sequence that resulted incleavage and secretion of the correct protein. The chloroplast genome ofa selected plant like tobacco can be readily transformed with auniversal vector as described herein and made to express HSA.General Applicability. As described herein the universal vector permits,in accordance with the invention, the transformation of plants, make theplant to express a biological molecule which can impart a desiredphenotype to the plant and/or produce a desired product which may, butneed not have biological activity (or a precursor to a final product).The coding nucleotide sequence can be synthetic, or natural. Theproduced molecule can be foreign to the plants, non-functional in theplant or functional. The universal vector has broad applications in thedomain of plant transformation.

It is contemplated that any biologically active molecule (precursor orderivative thereof) can be produced by transgenic plant transformed withthe universal vector of the invention, with suitable adaptations as maybe required for a particular case.

Herbicide Tolerance of Chloroplast Transformed Plants

Another embodiment of the invention relates to the use of the universalvector to confer herbicide resistance or tolerance to plants. Theadvancement of gene transfer technology has made possible theintroduction of herbicide resistance genes into plants, thereby makingthe herbicide selective for a particular crop.

Modifications of the Target Enzyme. The first step towards this approachis the identification and necessary modification of the target enzyme(gene) of the herbicide to confer tolerance, which has been performedquite extensively. Sulfometuron methyl is a sulfonylurea herbicide thatblocks the growth of bacteria, yeast, and higher plants by inhibitingthe first enzyme of the branched chain amino acids pathway, acetolactatesynthase (ALS). Mutant genes for ALS were isolated from E. coli andyeast that confer resistance to sulfometuron methyl. Yadav, et al.(1986). Herbicide resistance in tobacco, and several other crops hasbeen achieved by genetic engineering of the ALS gene. Gabard, et al.(1989); Miki, et al. (1990). Yet another approach to engineer herbicideresistance in plants has been the expression of the enzymephosphinothricin acetyl transferase (PAT) that detoxifies the herbicidephosphinothricin. DeBlock, et al. (1987).Glyphosate. Glyphosate is a potent, broad spectrum herbicide which ishighly effective against annual and perennial grasses and broad leafweeds. Glyphosate is environmentally safe as it rapidly degrades insoil, has minimal soil mobility, and has very little toxicity tonon-plant life forms. Glyphosate works by binding to and inhibiting theenzyme 5-enol-pyruvyl shikimate-3-phosphate (EPSP) synthase. EPSPsynthase (EPSPS) catalyzes the formation of EPSP, which is important inthe aromatic amino acid biosynthesis pathway, from shikimate-3-phosphateand inorganic phosphate. The non-toxicity of glyphosate to animals isdue to the fact that this reaction occurs only in plants andmicroorganisms. Unfortunately, because the reaction to form EPSP occursin all plants, glyphosate does not have selectivity between weeds anddesirable plants such as crops and ornamentals.

Two approaches have been used to attempt to develop a glyphosateresistant plant by genetic engineering. One approach is based uponoverproduction of wild type EPSP synthase, so that after competitiveinhibition of EPSP synthase by glyphosate, the residual EPSP synthaseconfers glyphosate tolerance. The second approach is based upon theexpression of a mutant gene (aroA) encoding glyphosate resistant EPSPsynthase.

In all of the aforementioned examples, without exception, herbicideresistant genes have been introduced into the nuclear genome.

The Need for Chloroplast Transformation. A serious need exists, todevelop a herbicide resistant plant, particularly a plant resistant tothe most widely used herbicides, in which the protein conferringherbicide resistance is produced in the chloroplast, and in which thegene conferring herbicide resistance cannot escape by pollen to theenvironment.

The universal vector of the invention responds to this need bytransformation of any target plant to provide tolerance to any selectedherbicide like to glyphosate. Important commercial crops like wheat,rice, corn (maize), soybean can be made resistant to a selectedherbicide by means of the universal vector.

The invention provides a transgenic herbicide resistant plant in which aforeign transgene conferring resistance to one or more herbicide isintegrated into the chloroplast genome by means of the universal vector.The transgenic plant may be a mature plant, an immature plant, such as aseedling, an embryo, a callus, a cultured tissue, or cell suspension, ora portion of a plant such as a cutting or a callus. Herbicides which aresuitable for the invention and for which genes conferring resistance maybe stably integrated into the chloroplast genome in accordance with theinvention include all known (and also to be developed) herbicides.

Class of Herbicides. Herbicides controllable by the invention have beengenerally grouped into several chemical classes.

A first class includes the PSII (Photosystem II) herbicides whichinterfere with the reduction of plastoquinone on the acceptor site ofPSII which include such chemicals as triazines, triazinones, ureaderivatives, biscarmates, nitrites, nitrophenols, substitutedpyridazinones, phenylcarbamates, anilides, cyanoacrylate, DCMU,carboanilides, uracils, specifically for example,dichlorophenyldimethylurea, atrazine, metribuzine, lenacil,phenmedipham, ioxynil and dinoseb. These chemicals bind to a 32-kDabinding protein (Q_(B) protein, D1, herbicide-binding protein) in thethylakoid membrane of chloroplasts, thereby blocking photosyntheticelectron transport. The plastid gene which codes for a precursor of theQ_(B) protein is named psbA, the sequences of which show a very highdegree of homology in different plants. The most important PSIIherbicide is atrazine, developed by Ciba-Gergy.

Another class are the PSI (Photosystem I) herbicides, a membrane boundprotein complex which catalyzes the light-driven oxidation ofplastocyanin (PC) and reduction of ferredoxin (Fd). Typical of thesechemicals are the bipyridyl herbicides paraquat and diquat.

Another class of herbicides are the aryloxyphenoxypropanoates (APP) oracetyl coenzyme A carboxylase type herbicides, which inhibits acetyl-CoAcarboxylase and subsequent fatty acid biosynthesis in plastids. Typicalof the APPs are cyclohexanedione (CHD), sethoxydin, haloxyfop,quizalofop, phenoxaprop (and the lower alkyl-substituted moleculesthereof), dicolofop, sethoxydin, clethodin and tralkoxydim.

Yet another class of herbicides includes the auxin analogs likemacropop, chloramben, dicamba, benazolin, 1-naphthylacetic acid;3,6-dichloropicolonic acid, picloram, fluoroxypyr, quinclorac, MCPA and2,4-D.

An additional class are the mitotic herbicides, termed dinitroanilineherbicides, like trifluralin, oryzalin and pendimethalin.

Another chemical class of herbicides to which the invention applies arethose acting in the biosynthesis of amino acids, such as tertiary theamino methyl phosphonic acids chlorsulfuron, glufosine and glyphosate.

Another class of herbicides are the acetolactate synthase-inhibitingherbicides (ALS), like the sulfonylureas, imidazolinones,triazolopyrimidines and pyrimidinyl thiobenzoates, such aschlorsulfuron, imazaphyr, flumetsulam (and others listed in chapter 4,Table I of Herbicide Resistance in Plants, 1994, cited below).

Examples of sulfonylurea herbicides are sulfometuron methyl (the activeingredient of Oust™) and chlorsulfuron (the active ingredient ofGlean™). Imazapyr, one of the imidazolinones, is the active ingredientof American Cyanamid's herbicide Arsenal™ and imazamethabenz is amixture of two imidazolinones (Merk Index, 11th Ed. 4825). Mutated formsof ALS located in the structural genes of ALS, ilvG and ILV2 may be usedto confer herbicide resistance using the universal vector.

In spite of the chemical differences between the imidazolinones andsulfonylureas, these substances inhibit the same enzyme, ALS. It appearsthat quinones and an imidazolinone herbicide compete with a sulfonylureaherbicide for a common site on ALS. Accordingly, in accordance with theinvention, plants can be transformed which will show a resistance toboth groups of these and other herbicides.

Another group of chemicals controllable by the invention using theuniversal vector which has herbicidal activity is typified byL-phosphinothricin, which is a component of the tripeptide “bialaphos”.This tripeptide is marketed under the trade name “Herbiace™” by MeijiSeika, Japan, and as “Basta™” by Hoechst AG, Germany. L-Phosphinothricinis a potent inhibitor of glutamine synthetase, which causes a rapidincrease of ammonia concentration plants and leads to the death of theplant cell.

In spite of extensive studies, no satisfactory product has beendeveloped to impart herbicide tolerance to plants by chloroplasttransformation. With the glyphosate type herbicide, it has been reportedthat regenerated nucleus transformed transgenic plants showed toleranceto glyphosate, but also showed impaired growth, and have not resulted intransgenic plants with high level of tolerance to glyphosate, Schultz,et al., 1990.

Chloroplast Transformants. In accordance with the invention, thechloroplast of target plants that are susceptible to herbicides istransformed with a vector of the invention carrying the necessary codingsequence, thereby conferring herbicide resistance. The transformedchloroplast comprises a genome which carries a foreign transgene whichconfers the herbicide tolerance. The chloroplast may be a maturechloroplast or may be an immature chloroplast, such as an etioplast.Preferably, the gene conferring herbicide resistance codes for EPSPsynthase which binds less readily (has reduced affinity) to glyphosatethan does wild type the EPSP synthase. If present, the second transgeneis generally a gene which confers antibiotic resistance to the plant.Thus, the herbicide resistance can be used by way of lethal selection asa marker for chloroplast transformation by selection of thetransformants by exposure to a medium with lethal concentration of theselected herbicide, to which the transformants will survive.

The origin of the second gene can be procaryotic (for example,bacterial) or eucaryotic (for example, plant).

The invention also relates to produce a plant resistant to severalherbicides, whether of the same class of herbicide or of differentclasses of herbicides, and the transformed plant resistant to multipleherbicides.

Some plant species are known to have a developed a natural andnon-permanent tolerance to certain types of herbicides. The teaching ofthis invention is readily applicable thereto.

The invention includes a method for producing a herbicide resistantplant which comprises transforming the chloroplast of the plant byintroducing one or more foreign transgenes which code for a proteinconferring herbicide resistance to the genome of the chloroplast of theplant. Preferably, the transgene codes for a mutant form of the enzymewhich has decreased affinity for a given herbicide than does thenaturally occurring enzyme.

The following Table lists a variety of type of resistance determinants,(chemicals or “molecules”) which inhibit or which confer resistance, andtypical herbicides related thereto.

TABLE I RESISTANCE DETERMINANT HERBICIDES glutathione S-transferases-triazine simazine chloracetamide metalachlor Auxin analogs 2,4-D MCPAmecopop chloramben EPSP synthase glyphosate Q_(b) (psbA) - PS II Typeatrazine terbutyne dichloropheny- dimethylurea metribuzine lenacilphenmedipham loxynil dinoseb Acetohydroxyacid sythase sulfonylureas(ALS) chlorosulfuron imazapyr sulfometuron methyl imidazolinonesGlutamine synthase phosphinothricin PS I Type paraquat diquat Acetylcoenzyme A carboxylase aryloxyphenoxypropanoate inhibiting enzymes (APPType) (APR), cyclohexanedione Mitotic disrupters trifluralin, oryzalin,pendimethalin, dinitroaniline

For a comprehensive review on the topic of herbicide resistance inplants, see Herbicide Resistance Crops, Agricultural, Environmental,Economic, Regulatory and Technical Aspects, 1996, CRC Press, Inc.,Editor, Stephen O. Duke and Herbicide Resistance in Plants, Biology andBiochemistry, 1994, CRC, Press, Edited by Stephen B. Powles and JosephA. M. Holtum. In the first of these reference books, Chapter 3 onTechniques for Producing Resistant Crops (and the numerous referencescited therein) is of particular interest as background to the invention.Both books are incorporated herein by reference in their entirety.

In accordance with the invention it has also been discovered that in theprocess for expressing a target trait another trait can be expressed bythe transformed plant which may be quite desirable, indeed may be moredesirable than the initially target trait. In those situations, theplants will be allowed to express and will be selected on the basis ofthat other trait.

The invention is exemplified in the following non-limiting examples.

EXAMPLE 1 Universal Chloroplast Integration and Expression Vectors

Tobacco. Exemplary universal chloroplast vectors were constructed byfirst cutting out the tobacco chloroplast DNA BamHI fragment(130656-140992) containing the 16S and 23S rRNA genes and subcloning itinto a commonly available bacterial plasmid pUC19. A map of the tobaccochloroplast genome is shown in FIG. 1. A 2.1 kbp HindIII-EcoRI fragmentpresent within this fragment, containing a universal border sequencecomprising trnI and trnA genes, including the spacer region between thegenes, was subcloned into the pUC19 plasmid at the PvuII site. Theresultant plasmid was designated pSBL-Ct Bor (FIG. 5).

The vector pSBL-RD-EPSPS (FIG. 2B) contains a mutant EPSP synthase genethat codes for the enzyme EPSP synthase. Glyphosate, the activeingredient in Mosanto's ROUND UP™, binds to the protein EPSP-synthaseand blocks the synthesis of essential amino acids, resulting in death ofa plant. The EPSP synthase coded for by the mutant gene does not bindglyphosate, and therefore confers herbicide resistance to crop plants.

Other genes, such as those that confer resistance to adverseenvironmental factors such as salt/drought tolerance (osmotolerancegenes such as betaine aldehyde dehydrogenase, (BADH), for theoverproduction of glycine betaine) or thermotolerance (genes coding forheat shock proteins) or cold shock tolerance proteins, or to pathogenresistance, such as antimicrobial (lytic peptides, chitinase) orantiviral (coat proteins) can be inserted singly or in non-conflictingcombinations into the universal chloroplast vector, or into differentcassettes of the same universal chloroplast vector to transform thetarget plant into one with the desired trait.

Construction of a Universal Chloroplast Integration Vector Containing aSynthetic Spacer 2 Region

A universal chloroplast vector containing only the spacer 2 region ofthe tobacco chloroplast genome was constructed by first subcloning asynthetic oligonucleotide comprising the spacer 2 region into thebacterial plasmid pUC19. The positive and negative strands of the 64base pair spacer sequence were synthesized, the sequence of the positivestrand was as follows:

5′-GCTGCGCCAGGGAAAAGAATAGAAGAAGCATCTGACTACTTCATGC ATGCTCCACTTGGCTCGG-3′SEQ ID NO: 5

The synthetic fragments were mixed and allowed to anneal, then ligatedinto pUC19 at the PvuII site. (FIG. 5B) Insertion of an appropriateselectable marker gene and a heterologous gene were as described abovefor pSBL-CtBor. (FIG. 5C)

To prepare a longer sequence which includes the tRNA^(Ile) and thetRNA^(Ala) genes, the same methodology is followed.

Transformation of Different Plants EXAMPLE 2

Chloroplast Transformation of Tobacco. The following example describes aclassic protocol for transformation of tobacco chloroplast for which anyvector can be used. Two such vectors are identified below. All newchloroplast vectors were first tested in tobacco as described inDaniell, (1997). Tobacco (Nicotiana tabacum var. Petit Havana) plantswere grown aseptically by germination of seeds on MSO medium containingMS salts (4.3 g/liter), B5 vitamin mixture (myo-inositol, 100 mg/liter;thiamine-HCl, 10 mg/liter; nicotinic acid, 1 mg/liter; pyridoxine-HCl, 1mg/liter), sucrose (30 g/liter) and phytagar (6 g/liter) at pH 5.8.Fully expanded green leaves of about two month old plants were selectedfor bombardment.

Leaves were placed abaxial side up on a Whatman No. 1 filter paperlaying on RMOP* medium in standard Petri plates (100×15 mm) forbombardment. Tungsten (1 μm) or Gold (0.6 μm) microprojectiles werecoated with plasmid DNA, of interest (e.g. PSBL-RD-EPSPS orpZS-RD-EPSPS) and bombardments were carried out with the biolisticdevice PDS1000/He (Bio-Rad) as described by Daniell, 1997. Followingbombardment, petri plates were sealed with parafilm and incubated at 24°C. under 12 h photoperiod. Two days after bombardment, leaves werechopped into small pieces of about 5 mm² in size and placed on thelethal selection medium (RMOP containing a selectable marker such asabout 500 μg/ml of spectinomycin dihydrochloride) with abaxial sidetouching the medium in deep (100×25 mm) petri plates (about 10 piecesper plate). Selected from the shoots that died, the regeneratedspectinomycin resistant shoots were chopped into small pieces (about 2mm²) and subcloned into fresh deep petri plates (about 5 pieces perplate) containing the same lethal selection medium. Resistant shootsfrom the second culture cycle were transferred to rooting medium (MSOmedium supplemented with IBA, 1 Ag/liter and an appropriate antibioticlike 500 μg/ml of spectinomycin dihydrochloride). Rooted plants weretransferred to soil and grown at 26° C. under continuous lightingconditions for further analysis.

After transfer to the lethal selection medium, the explants graduallybecame pale and in about 3-8 weeks, green calli and shoots developedfrom the bombarded side of the 0leaf. Resistant shoots from each calluswere considered as a clone.

PCR screening for chloroplast transformants after the first culturecycle showed that 12 out of 20 resistant clones integrate the foreigngenes like the aadA gene linked to the EG121 gene into the chloroplastgenome. These 12 clones were advanced to further steps of regeneration.The entire process of regeneration, starting from bombardment untiltransfer to soil, takes about 3-5 months.

FIG. 9 shows transformed and untransformed tobacco plastids growing inthe presence of spectinomycin indicating non-lethal selection on themedium (500 μg/ml).

EXAMPLE 3

Corn Chloroplast Transformation. Surface sterilization and germinationof corn seeds. Corn seeds are surface sterilized in a solutioncontaining 20′ (v/v) commercial bleach and 0.5% SDS for 15 min undercontinuous shaking, then serially rinsed in sterile double-distilledwater (sddw) four to five times. Liquid MS-based germination medium(modified CSG) containing MS salts (4.3 g/l), sucrose (30 g/l),DM-vitamins (1.0 mg/l thiamine-HCl, 0.5 mg/l nicotinic acid, 0.5 mg/lpyridoxine-HCl and 100 mg/l myo-inositol) and BA (2.0 mg/l) at pH 5.8 isdispensed per Magenta™ box (45 ml) containing eight layers ofcheesecloth, then autoclaved. Seeds are placed in modified CSG (25 seedsof any genotype per box) and cultured for three days (16 h or continuouslight; 25 C) for germination. Nodal sections are excised asepticallyfrom three day-old seedlings. The nodal section appears as a cleardemarcation on the germinating seedling and represents the seventh node(FIG. 10A). When excised, the nodal cross-sections are approximately1.2-1.5 mm in length (FIG. 10B).

FIGS. 10A-G shows corn plastid transformation and regeneration scheme.A) Three day-old corn seedling; arrows and line depict the seventh nodefor explant excission; B) Nodal cross sections prior to bombardment;arrows depict margin of one section; C) GUS-positive nodal section(nuclear transformation); histochemical assay conducted three dayspost-bombardment; D) Multiple shoot induction from one nodal section(control) after eight weeks in culture; E) Control shoot on elongationmedium for three weeks; F) Rooted control plantlet; G) Selection ofplastid transformed corn in liquid medium containing spectinomycin andstreptomycin for eight weeks.

Multiple shoot induction: Nodal section explants are placed on cornshoot induction medium [CSI; MS salts, sucrose and DM-vitamins as above,BA (2.0 mg/l), CPA (0.25 mg/l) and phytagar (8 g/l) at pH 5.8],acropetal end up, and placed under the culture conditions previouslymentioned. In all media except modified CSG and RG1 (rice), PGRs andantibiotics are filter-sterilized and added after autoclaving. Tissuesare subcultured every two weeks onto fresh CSI medium for multiple shootformation (FIG. 10D). Adventitious shoots are separated from the shootclumps after eight weeks of culture and elongated on semi-solid MS-basedmedium containing sucrose, DM-vitamins, glycine (10 mg/l) and asparagine(150 mg/l) at pH 5.8, for three weeks (FIG. 10E). The plantlets arerooted (FIG. 10F) on the same medium containing IBA (0.5 mg/l). Rootedplantlets can be grown in PGR-free liquid MS in test tubes (150×25 mm)containing cheesecloth as the anchor material to achieve faster growth.Regenerated plantlets are transplanted to potting media, acclimatizedthen grown to maturity in the greenhouse.

FIG. 11 shows corn plastid transformation. Transformed corn plants grownormally (middle shoot) while untransformed plants die on the lethalmedium, confirming lethal selection by the antibiotic spectinomycin(1000 μg/ml).

EXAMPLE 4

Rice Chloroplast Transformation. Surface sterilization of rice seeds andpreculture. Dehusked seeds from any genotype (indica or japonica types)are surface sterilized first in 70% ethanol for 10 min under continuousshaking then rinsed with ddw about five times. Seeds are then soaked ina 0.2% Benlate (w/v) solution for 20 min, rinsed with sddw five times,then in 50% bleach for 20 min with the sddw rinses repeated. Seeds arepre-cultured in medium RG1 [MS salts, sucrose and DM-vitamins as above,BA (2.0 mg/l) at pH 5.8]. As with corn, liquid RG1 is dispensed toMagenta™ boxes containing cheesecloth prior to autoclaving. Seeds areplaced in RG1 (100 seeds of any genotype per box) and pre-culturedovernight (16 h or continuous light; 23 C) prior to bombardment thefollowing day.Bombardment of embryos on intact rice seeds. Pre-cultured seeds aretightly packed vertically, embryo end up (FIG. 12A), in the central 2.5cm area of a petri dish (25 per dish) containing medium RG1.1 (RG1 plus8.0 g/l phytagar) and bombarded with DNA-coated microprojectiles.DNA precipitation. The procedure is as described for corn with thefollowing modifications. Ten μl DNA (1.0 μg/μl) and 20 μl isopropanol(2×vol of DNA), 60 μl 2.5 M CaCl₂ and 15 μl 0.1M spermidine are used.Each shot delivers 2.0 μg DNA and 720 μg tungsten.

FIGS. 12A-F shows rice plastid transformation and regeneration scheme.A) Rice seeds, embryo end up, just prior to bombardment; arrows point toembryo margins; B) Multiple shoot induction from one control embryoafter seven weeks in culture; C) Selection of plastid transformed riceshoots arising from one initial rice embryo in media containingspectinomycin and streptomycin after eight weeks in selective medium;arrows point to two putative transformants; D) Control rice regenerants;E) Transgenic Priscilla 2.3; F) Transgenic Priscilla 2.4.

Multiple shoot induction and selection of transplastomicspost-bombardment. The rice seeds are separated and spread out on theRG1.1 medium (maintaining the polarity) and placed in the dark for twodays post-bombardment. The embryo end of the seed is then cut away(embryo plus small amount of endosperm) from the remainder of endospermwhich is discarded. Embryos are placed in 50 ml liquid RG2 medium (in250 ml flask) for multiple shoot induction (FIG. 12B). RG2 contains MSsalts, sucrose and DM-vitamins as above, and BA (6.0 mg/l) at pH 5.8.RG2 used for selection includes spectinomycin (1000 μg/ml) plusstreptomycin sulfate (100 μg/ml). The cultures are placed in the growthchamber (16 h photoperiod; 25° C.) and subcultured every two weeks intofresh selection media. Green shoots are selected from the shoot clumpsarising from each embryo and placed back in selective media (FIG. 12C).Rooting is achieved in medium RG3 [MS salts, sucrose and DM-vitamins asabove, IBA (0.5 mg/l) at pH 5.8] plus antibiotics. (Shoots can be eitherrooted separately or as multiple shoot clusters). Plantlets aretransplanted to potting media, acclimatized then repotted to a clay:sand(1:1) mix and grown to maturity in the greenhouse. (FIGS. 12D,E,F).Development of plastid transformation and regeneration protocols forcorn and rice. As described above unique corn nuclear transformation andregeneration protocols (FIG. 10) were developed (Rudraswamy, 1997) andadapted for plastid transformation. (Previous to this work, nodalsection explants had not been used for transformation or regeneration.)Multiple shoots were induced on nodal sections excised from threeday-old seedlings of 21 genotypes (none related to A188 or B73) whichincluded hybrid (16 grain, one sweet) and inbred (four) genotypes. Aftereight weeks in culture, 16-32 shoots (avg. 24) were generated perexplant. Shoots were rooted and regenerants did not display aberrantphenotypes in greenhouse analyses (limited study of two plants pergenotype). DNA could also be delivered into nodal section explants ofall genotypes (FIG. 10C; transient β-glucuronidase expression). Forplastid transformation, nodal section explants were bombarded withpSBL-ctV2, then placed on a multiple shoot induction medium containingspectinomycin and streptomycin. Arising shoots could be excised andre-placed on shoot induction medium for subsequent rounds of selection.

As described above unique rice targets dehusked intact mature seeds,embryo end up, not used in previously reported transformation protocolswere coupled with a multiple shoot induction protocol (FIG. 12) formature embryos (excised two days post-bombardment). Multiple shoots wereinduced on all eight genotypes tested (Litton, Priscilla—two newlyreleased Mississippi cultivars, plus six breeding lines). The notedresponse should be similar in numerous other cultivars since the initialexplant is a mature embryo. Regenerants (non-transformed) are beingmaintained for collection of F1 seed. After plastid transformation,shoot multiplication occurred in the presence ofspectinomycin/streptomycin and, as with corn, shoots could undergonumerous rounds of selection due to shoot proliferation (unknown ifaxillary or adventitious in origin) from the base of excised shoots.Rooting was also accomplished in selective media.

FIGS. 13A-B shows PCR analysis of DNA isolated from first generationleaves of rice transformants. PCR analysis was done with DNA isolatedfrom the first generation leaves. The PCR products were not abundant asobserved in tobacco chloroplast transgenic plants (FIGS. 13A, lane 11,13B, lane 12). This may be because of two reasons. The protocol used toisolate DNA is not suitable for coarse rice leaves or that the primersdesigned for tobacco do not anneal as well with rice ct DNA.Nevertheless, for preliminary analysis, tobacco primers were used totest integration of the aadA gene into the plant genome from theuniversal vector. Lack of a product would indicate spontaneous mutants,capable of growing on spectinomycin without the aadA gene (FIG. 13A,lanes 7-10). A PCR product of 1.57 Kb was detected in four lines (FIG.13A, lanes 2-6) transformed with the universal vector. Under theselection conditions used, four mutants were detected out of ten linestransformed with the universal vector. Primers were also designed tospecifically identify integration into the plastid genome. For theuniversal vector, the primer on the native chloroplast genome landed inthe 16s rRNA gene, outside the flanking sequence of the chloroplastvector (1.60 Kb PCR product). The expected products were observed forthe transgenic lines obtained using the universal vector (FIG. 13B,lanes 5, 6). Unbombarded plants (controls) did not yield any PCRproducts, as expected (FIG. 13B, lane 2; FIG. 13A, lane 1). PCR resultsidentified two ‘Priscilla’ rice plants (2.3 and 2.4) which containtransformed plastids (FIGS. 12 and 13).

EXAMPLE 5 Peanut Chloroplast Transformation Arachis hypogaea

Transgenic peanuts having transformed chloroplast genomes were obtainedusing the universal vector pSBL-CG-CtV2 (FIG. 7A). Peanut tissue wasgrown in culture in accordance with the protocol described by Kanyand etal., 1994. Bombardment conditions were the same as for tobaccochloroplast transformation as described above, except that epicotylsections were used for bombardment while using rupture discs of variablepressure. Peanut chloroplast transformation has never been previouslyreported.

FIG. 14 shows peanut plastid transformation. Transformed peanut plantsgrow normally (middle and on left side of plate) while untransformedplants die in the lethal medium (500 μg/ml).

EXAMPLE 6

Soybean Chloroplast Transformation. Transgenic soybeans havingtransformed chloroplast genomes were obtained using the universal vectorpSBL-CG-CtV2 (FIG. 7A). Bombardment conditions were as for tobaccochloroplast transformation. Soybean chloroplast transformation has neverbeen previously reported.

FIG. 15 shows soybean plastic transformation. Two transformed plantsshow shoots, the other plant die on the lethal medium, confirming lethalselection by the antibiotic spectinomycin (500 μg/ml).

EXAMPLE 7

Sweet Potato Chloroplast Transformation. Transgenic sweet potato plantshaving transformed chloroplast genomes were obtained using the universalvector pSBL-CG-CtV2 (FIG. 7A). Sweet potato tissue were grown in culturein accordance with the protocol described by Zhang et al., 1996.Bombardment conditions were the same as for tobacco chloroplasttransformation as described above, except that calli and primary embryoswere bombarded and, after bombardment, were transferred to platescontaining 100 mg/ml spectinomycin. Sweet potato chloroplasttransformation has never been previously reported.

FIG. 16 shows sweet potato embryos transformation on the lethalantibiotic spectinomycin selection medium (500 μg/ml). Note bleachedcalli (right) and green embryos (left).

EXAMPLE 8

Grape Chloroplast Transformation. Transgenic grape plants havingtransformed chloroplast genomes are obtained using the same universalvector pSBL-CG-CtV2. Grape tissue are grown in culture according to theprotocol of Hebert et al., 1993. All chloroplast transformationprotocols are as for tobacco, except that cells in the exponential phaseof growth, about 4 days after subculturing, were used for bombardment.Grape chloroplast transformation has never been previously reported.

FIG. 17 shows grape cells transformation. The transformed culture cellsbecome green while the untransformed cells die in the lethal antibioticspectinomycin selection medium (500 μg/ml).

EXAMPLE 9

Transformation of Other Plants. Transformation of plants by means ofmicroprojectile bombardment is a favored technique to introduce theuniversal vector carrying the desired nucleotide sequence coding for themolecule of interest into the target plants. Illustrative transgenicplants obtained through microprojectile bombardment are shown in TableII.

TABLE II Transgenic Plants Recovered Through Microprojectile BombardmentPlant Species Explant Utilized for Transformation Alfalfa Cali frompetiole, stem sections Arabidopsis Root sections Barley Embryogeniccallus, immature embryos Banana Embryogenic suspension cells BeanMeristems Citrus Embryogenic cells Cotton Embryogenic suspensions;meristems Cranberry Stem sections Cucumber Cotyledons Dendrobium orchidProtocorms Eucalyptus Zygotic embryos Grape Embryogenic suspension cellsMaize Embryogenic suspensions; immature embryos Oat Embryogenic PapayaZygotic/somatic embryos; hypocotyls Pasture grass Embryogenic calliPeach Embryo derived calli Peanut Meristems Poplar Embryogenic RiceZygotic embryos

The transformation of plants by the use of the gene gun is described inDaniell, 1997. Each crop that was reported to be nuclear transformablevia microprojectile bombardment in that Table can have its chloroplastgenome transformed using the universal vector as described herein.

EXAMPLE 10 Expression of Non-Plant Products

The examples that follow, illustrate the expression of biodegradableprotein-based biopolymers (PEPs) and analysis of transformants.

Vector pSBL-CG-EG121. The vector pSBL-CG-EG121 (FIG. 3A) contains thegene (GVGVP)_(121mer) (designated EG121) which codes for a biodegradableprotein-based biopolymer (PBP) that has many medical and non-medicalapplications.

Construction of Chloroplast Expression Vectors. Standard protocols forvector construction were as outlined by Sambrook et al., 1989.Chloroplast integration and expression vectors pSBL-CtV2 (FIG. 7A) andpZS197 were digested, respectively, with XbaI (an unique site betweenthe aadA gene and the psbA 3′ region) and SpeI (a unique site at 120 bpdownstream of the aadA geen in the psbA 3′ regulatory region), Klenowfilled and dephosphorylated. The polymer gene EG121 along with theShine-Dalgarno sequence (GAAGGAG) from the pET11d vector was excised asa XbaI-BamHI fragment from the plasmid pET11d-EG121. Sticky ends of theinsert fragment were Klenow filled and ligated with vectors pSBL-CtV2 orpZS197 yielding chloroplast expression vectors pSBL-CG-EG121 (FIG. 3A)and pZS-CG-EG121 (FIG. 3B), which integrate the aadA and EG121 genes atthe inverted repeat (IR) or into the spacer region between the rbcl andorf 512 genes of the tobacco chloroplast genome. Refer to FIG. 1, for“V” and “TV”, integration sites, respectively.Biopolymer Expression in E. coli and Tobacco. Plasmid vectorpSBL-CG-EG121 (FIG. 3A) was transformed into E. coli strain XL-1 Blueand grown in Terrific Broth in the presence of ampicillin (100 μg/ml) at37° C. for 24 h. SDS-PAGE was carried out according to Laemmli, 1970using a 12% resolving gel and a 5% stacking gel and run for 5 h at aconstant current of 30 mAmps. Crude protein extracts from E. coli cellswere prepared and electrophoresed as described by Guda et al., 1995.After electrophoresis, polypeptides were visualized by negative stainingwith CuCl₂.

FIG. 18 shows expression of chloroplast integration and expressionvectors in E. coli strain HMS174 (DE3). Lane 1, shows the purifiedpolymer protein; lane 2, shows the untransformed E. coli control; lane3, shows the E. coli strain XL-1 Blue transformed with universal vectorPSBL-CG-EG121 (FIG. 3A); lane 4 shows E. coli transformed with thetobacco vector and lane 5, shows E. coli strain HMS174 (DE3) transformedwith pET11d-EG121 vector, in which the T7 promoter transcribes thepolymer gene. The level of expression by the Prrn promoter in E. coliwas almost equivalent to that of the highly efficient T7 promoterdriving the polymer gene.

Southern Blot Analysis. Total DNA was extracted from leaves oftransformed and wild type plants using the CTAB procedure of Rogers andBendich, 1988.

FIGS. 19 and 20 show Southern blot analysis performed independently withthe transformants obtained using the tobacco (FIG. 19) and the universal(FIG. 20) vectors. Total DNA was digested with EcoRI and HindIII in caseof the universal vector (UV) transformants or EcoRI and EcoRV in case ofthe tobacco vector (TV) transformants. Presence of an EcoRI site at the3′ end of the polymer gene allowed excision of predicted size fragmentsin the chloroplast transformants only. To confirm foreign geneintegration and homoplasmy, individual blots were probed withcorresponding border sequences. In the case of the TV transformantsafter the second or third round of selection, the border sequencehybridized with 4.6 and 1.6 kbp fragments (FIG. 19A, lanes 2, 3 and 4)and with a 3.1 kbp native fragment in the wild type (FIG. 19A, lane 1).On the other hand, in the case of the UV transformants, after the firstround of selection, the border sequence hybridized with 4.0 kbp and 1.2kbp fragments (FIG. 20A, lanes 1 and 2) while it hybridized with anative 2.1 kbp fragment in the control (FIG. 20A, lane 3). Moreover, TVtransformants also showed the native fragment of 3.1 kbp (FIG. 19A,lanes 2 and 3) similar to the wild type plant indicating heteroplasmiccondition of the transformed chloroplast genomes, even though they havebeen under several rounds of selection. However, both UV transformantsshowed homoplasmic condition, even after the first round of selection(FIG. 20A, lanes 1, 2).

Presence of heteroplasmy even after second selection was reportedearlier and it was suggested that selection should be done untilattainment of homoplasmy (Svab and Maliga, 1993). This is consistentwith the observation that a high degree of heteroplasmy exists after asecond selection cycle in the TV transformants (FIG. 19A, lanes 2 and3). However, no heteroplasmic condition was observed in case of the UVtransformants which may be because of the copy correction mechanismbetween the two IR regions and/or the presence of chloroplast origin ofreplication (ori) within the border sequence, which should increase thecopy number of the introduced plasmid before integration.

DNA gel blots were also probed with either the aadA gene (UV integratedplants) or the EG121 gene (TV integrated plants) to reconfirmintegration of foreign genes into the chloroplast genomes. In the TVintegrated plants, the polymer gene probe hybridized with a 4.6 kbpfragment, only in the plastid transformant lines (FIG. 19B, lanes 2, 3and 4). Also, in the UV integrated plants, aadA sequence hybridized withan expected 4.0 kbp fragment (FIG. 20B) which also hybridized with theborder sequence in plastid transformant lines (FIG. 20A, lanes 1 and 2).

Analysis of Transcript Levels in Transgenic Tobacco and Northern Blot.Foreign gene transcript levels were analyzed by northern blotting (FIG.21) using total RNA isolated from the control, chloroplast transformantsand a tobacco transgenic plant highly expressing the polymer gene(EG121) via the nuclear genome (Zhang et al., 1996). The polymer gene(EG121) sequence hybridized with a 1.8 kbp fragment in the chloroplasttransformants (lanes 1-4, 5-6) and also with larger size fragments inone of the chloroplast transformants (lane 6). In the case of thenuclear transformant, a transcript of about 2.1 kbp was observed (lane7). This was due to the presence of a poly A tail at the 3′ end of thepolymer transcript provided by the nos terminator. The larger sizefragments observed in lane 6 may be the di-, tri- or polycistronictranscripts which are being processed in chloroplasts. This is a commonphenomenon in chloroplast gene expression because many plastid genes areorganized into polycistronic transcriptional units that give rise tocomplex sets of overlapping mRNAs. Quantitation of transcript levelsrevealed that the chloroplast transformants were producing a 11-fold(lane 5) or more than fifty fold (lane 6) of polymer transcripts overthat of a highly expressing nuclear transformant (lane 7, highestexpressing plant among thirty five TV nuclear transgenic plantsexamined). This is directly attributed to the presence of higher genecopy numbers in chloroplasts of transgenic plants. The tobacco vector(TV) integrated plastid transformants showed lower levels of polymertranscript (lanes 1-4) compared to the universal vector integratedtransformants (lanes 5, 6) because the polymer gene exists as two copiesper transformed plastid genome in universal vector transformants asagainst a single copy in the TV transformants and the heteroplasmicconditions observed in TV transformants.Western Blot Analysis. Polymer protein was purified from leaves fromwild type tobacco, chloroplast transformants and nuclear transgenicplants following the method recently described by Zhang et al., 1995.Purified polymer was analyzed by SDS-PAGE according to Laemmli, 1970using a 120 resolving gel and a 5% stacking gel and run for 5 h at aconstant current of 30 mAmps. Polymer polypeptides of about 60 kDa werevisualized by negative staining with 0.3 M CuCl₂. Gels were destained in0.25 M sodium EDTA and 0.25 M Tris-Cl, pH 9.0 with three changes ofbuffer at 10 min intervals. Western immunoblotting and staining (FIG.22) was carried out as described by Zhang et al., 1996 using amonoclonal antiserum raised against the polymer AVGVP which cross-reactswell with polymer GVGVP and the “Immuno-Blot Assay Kit” (Bio-Rad). Thepolymer polypeptides running at about 60 kDa are seen in the plastidtransformants of IR integrated plants. Polymer expression from a highlyexpressing F2 generation nuclear transgenic plant (highest expressingplant among 35 transgenic plants examined) is seen in lane 5 (FIG. 22),while no polymer was expressed in the untransformed control as seen inlane 4 (FIG. 22). Eleven to fifty fold higher level of polymertranscripts is shown in the chloroplast transformants (FIG. 21). In thecase of chloroplast native occurring proteins like valine and prolinewhose biosynthetic pathways are compartmentalized in chloroplasts,higher levels of protein can be expected to be produced.

EXAMPLE 11 Genetic Engineering for Glyphosate Tolerance Via the Nuclearand Chloroplast Genomes

Chloroplast Integration and Expression Vectors with EPSPS in Tobacco.The EPSPS coding sequence has been recently integrated into the tobaccochloroplast genome (FIG. 1). The chloroplast vector PZS-RD-EPSPS (FIG.2A) contains the 16S rRNA promoter (Prrn) driving the aadA and EPSPSgenes with the psbA 3′ region from the tobacco chloroplast genome. Thisconstruct integrates the EPSPS and aadA genes into the spacer regionbetween the rbcL and orf512 genes of the tobacco chloroplast genome.FIG. 1, at the “TV” arrow.Test for Glyphosate Resistance. Gene Expression in E. coli. Because ofthe high similarity in the transcription and translation systems betweenE. coli and chloroplasts (Brixey et al., 1997), chloroplast expressionvectors were first tested in E. coli for resistance in this case toglyphosate before proceeding with transformation of higher plants. Thehigher growth rate of E. coli containing the tobacco vector compared tothe control containing pZS197 (similar to pZS-RD-EPSPS but lacked theEPSPS gene) in the presence of 10 mM and 40 mM glyphosate (FIG. 23A)indicates glyphosate tolerance of E. coli expressing the EPSPS gene.Another growth curve (FIG. 23B), confirms the expression of EPSPS viathe universal vector in E. coli. Thus, glyphosate tolerance of E. coliis due to the expression of the EPSPS gene, present in both the tobaccoand universal vectors.

Characterization of Tobacco Transgenic Plants

Integration of the Gene. Fully expanded green leaves of Nicotianatabaccum var. Petit Havana were bombarded with the tobacco and theuniversal chloroplast vectors. Two days after bombardment, leaf explantswere transferred to selection lethal medium containing spectinomycin(500 μg/ml). Transgenic plants were obtained within 3-5 months afterbombardment. Typically, out of 16 bombarded leaves, 10 independentlytransformed shoots were identified.

PCR analysis was performed with DNA isolated from the first or secondgeneration shoots and also from the mature transgenic plants. Primerswere used to confirm integration of the aadA gene into the plant genomefrom the tobacco as well as universal vectors. Lack of a product wouldindicate spontaneous mutants, capable of growing on spectinomycinwithout the aadA gene. The expected PCR product (887 bp) was obtainedfrom six lines (FIGS. 24A-B, lanes 1-6) transformed with the tobaccovector. A PCR product of 1.57 Kb was detected in four lines (FIGS.24A-B, lanes 1-4) transformed with the universal vector. Under theselection conditions used, four mutants were detected out of ten linestransformed with the tobacco vector. On the other hand, all thetransgenic lines transformed with the universal vector showedintegration of the aadA gene.

PCR Chloroplast Integration. Primers were also designed to specificallyidentify integration into the plastid genome. The strategy here was toland one primer on the native chloroplast genome, adjacent to the pointof integration of the vector, while landing the other on the aadA gene.A primer was designed to land immediately outside the rbcL gene in thetobacco vector (2.08 Kb PCR product). For the universal vector, theprimer on the native chloroplast genome landed in the 16s rRNA gene(1.60 Kb PCR product). The expected products were observed for thetransgenic lines obtained using the tobacco vector (FIGS. 24A-B, lanes2-7) as well as the universal vector (FIGS. 24A-B, lanes 1-4).Unbombarded plants (controls) did not yield any PCR products, asexpected (FIGS. 24A, lanes 1 and 9; 24B, lanes 5 and 11). Thus, alltransgenic plants examined turned out to be chloroplast and not nucleartransformants perhaps due to the requirement of higher levels ofaminoglycoside adenyl transferase (AADA) in transgenic plants understringent selection conditions. Low levels of AADA present in thecodicil of nuclear transgenic plants, should have eliminated nucleartransformants. The results of PCR analysis are conclusive and providedefinitive evidence for chloroplast integration of foreign genes usingboth the tobacco and universal vectors.Southern Analysis. The integration of the aroA gene into the chloroplastwas also confirmed by Southern analysis. In addition, the high level ofresistance to glyphosate observed (FIG. 20A) was confirmed bydetermination of the copy number of the foreign gene in the transgenicplants. The probe, to determine integration of the foreign gene into thechloroplast genome, comprised 654 bp fragment of the EPSPS gene, randomprimer labeled with P³². The total DNA comprising both organellar andgenomic was digested with EcoRI. The presence of an EcoRI site 200 bpupstream from the integration site, in the chloroplast genome, was usedto confirm integration of the EPSPS gene into the chloroplast genome intransgenic plants. The probe hybridized to the native EPSPS gene,present in the nuclear genome, is seen as 4.5 Kb fragment. In addition,the probe hybridized to the digested chloroplast genomes of thetransgenic tobacco plants, generating the 3.5 kb and 4.35 Kb fragmentsin FIG. 25A, lanes 2, 3 and 4. The probe did not hybridize to thedigested chloroplast genome of the untransformed control plant (FIG.25A, lane 1) since the foreign gene is not present in the chloroplastgenome of tobacco. This clearly establishes the integration, of theEPSPS gene, into the chloroplast genome.Gene Copy Numbers. The copy number of the integrated gene was determinedby establishing homoplasmy for the transgenic chloroplast genome.Tobacco chloroplasts contain 5000-10,000 copies of their genome percell. (McBride et al, 1995) If only a fraction of the genomes areactually transformed, the copy number, by default, must be less than10,000. By establishing that in the transgenics the EPSPS transformedgenome is the only one present, one could establish that the copy numberis 5000-10,000 per cell. This was shown by digesting the total DNA withEcoRI and probing, with the flanking sequences that enable homologousrecombination into the chloroplast genome. The probe comprised a 2.9 Kbfragment of the rbcL-orf 512 sequences. A chloroplast genome transformedwith the EPSPS gene, incorporates an EcoRI site between the rbcL-orf 512region of the chloroplast genome, thereby generating an extra fragmentwhen digested with this enzyme (FIG. 25C). Southern hybridizationanalysis revealed a 4.43 Kb fragment in FIG. 25B, lane 1 for theuntransformed control. In lanes 2, 3 and 4, two fragments (4.35 Kb and 3Kb) were generated due to the incorporation of the EPSPS gene cassettebetween the rbcL and orf512 regions (FIG. 25C provides a schematicdiagram with the dotted lines in gray signifying the point ofintegration of the foreign DNA). The 4.43 Kb fragment present in thecontrol is absent in the transgenics. This proves that only thetransgenic chloroplast genome is present in the cell and there is nonative, untransformed, chloroplast genome, without the EPSPS genepresent. This establishes the homoplasmic nature of the transformants,simultaneously providing an estimate of 5000-10,000 copies of theforeign EPSPS gene per cell. This would then explain the high levels oftolerance of glyphosate that was observed in the transgenic tobaccoplants (FIG. 20A).Progeny. Seeds collected from self-pollinated transgenic plants weregerminated in the presence of spectinomycin (500 μg/ml). All seedsgerminated, remained green and grew normally (FIG. 26B). Uniformspectinomycin resistance indicated that the aadA gene was transmitted toall progeny. Lack of variegation suggested homoplasmy because aheteroplasmic condition would have given rise to variegated progeny onspectinomycin (Svab et al., 1990; Svab and Maliga, 1993). The lack ofvariation in chlorophyll pigmentation among the progeny also underscoresthe absence of position effect, an artifact of nuclear transformation.All control seedlings are bleached, and did not grow in the presence ofspectinomycin (FIG. 26A).Tolerance of Glyphosate. Eighteen week old control and transgenic plantswere sprayed with equal volumes of glyphosate at differentconcentrations (0.5 to 5 mM). Control tobacco plants were extremelysensitive to glyphosate; they died within seven days even at 0.5 mMglyphosate (FIG. 27B). On the other hand, the chloroplast transgenicplants survived concentrations as high as 5 mM glyphosate (FIG. 27A).These results are intriguing, considering the fact that the EPSPS genefrom petunia used in these chloroplast vectors has a low level oftolerance to glyphosate and also contains the transit peptide fortargeting into chloroplasts.

This is the first report of a eukaryotic nuclear gene expression withinthe prokaryotic chloroplast compartment. It is well known that the codonpreference is significantly different between the prokaryoticchloroplast compartment and the eukaryotic nuclear compartment. Ideally,a mutant aroA gene (which does not bind glyphosate) from a prokaryoticsystem should be expressed in the chloroplast compartment. Such genesare now available and exhibit a thousand fold higher level of resistanceto glyphosate than the petunia gene used in this work. In light of theseobservations, it is possible that integration of prokaryotic herbicideresistance genes into the chloroplast genome as performed herein canresult in incredibly high levels of resistance to herbicides while stillmaintaining the efficacy of biological containment, i.e., avoiddissemination by pollen.

EXAMPLE 12

Tolerance of Corn to Glyphosate. A universal chloroplast vector usingcorn chloroplast DNA is constructed as follows. First, vectorpSBL-Ct-bor (FIG. 5C) is constructed as follows: Corn chloroplast DNAsubclone containing one of the inverted repeat regions is constructedwith bacterial plasmid pUC19. Second, a smaller subclone containing onlythe rRNA operon is constructed from the first subclone and the fragmentpresent in the second subclone containing the trnA and trnI genes andspacer regions representing the universal border are subcloned into apUC19 plasmid at the PvuII site. The resultant plasmid is designatedpSBL-Ct-bor. Within plasmid pSBL-Ct-bor, a selectable maker genecassette containing a chloroplast 16S rRNA promoter, the aadA gene(encoding aminoglycoside 3′adenyl transferase conferring resistance forstreptomycin/spectinomycin) and a 3′ untranslated region of thechloroplast psbA gene is inserted to construct vector pSBL-CORN. Theselectable maker gene cassette is inserted between the trnI and trnAgenes in the spacer region, in the direction of the 16S rDNAtranscription.

The vector pSBL-CORN-aroA, which contains a mutant aroA gene fromSalmonella typhimurium (Stalker et al. 1985; Comai et al. 1983) thatencodes the enzyme EPSPS synthase, is constructed by inserting themutant aroA gene into the pSBL-CORN vector. Transgenic corn plantsexpressing the mutant aroA gene are resistant to glyphosate treatmentlike “Roundup™” whereas the untransformed control plants are not.

EXAMPLE 13

Chloroplast Transformation for Tolerance to Imidizolinones orSulfonylureas. Plasmid pSBL-CORN is modified by insertion of a DNAfragment containing a mutated form of the acetolactate synthase gene ofSaccharomyces cerevisiae (Falco and Dumas. 1985; Yadav et al. 1986) togenerate plasmid pSBL-CORN-ASL1. This gene encodes an acetolactatesynthase that is not inhibited by imidizolinones or sulfonylureas andconfers tolerance to herbicides containing sulfometuron methyl andherbicides containing Imazapyr. Transformed tobacco plants expressingthe mutant acetolactate synthase gene are resistant to imidizolinone andsulfonylurea herbicide sprays.

The vector pSBL-CORN-ALS2 is a derivative of pSBL-CORN that containsmutated copies of the tobacco suRA and suRB genes (Chaleff and Ray.1984). These genes encode the tobacco acetolactate synthase polypeptide.The plasmid pSBL-CORN-ALS2 is constructed by ligating the suRA and suRBgenes, isolated from tobacco genomic DNA into the PSBL-CORN vector. Theresulting vector confers resistance to imidazolinone and sulfonylureaherbicides.

EXAMPLE 14

Chloroplast Transformation for Tolerance to Photosystem II Inhibitors.Photosystem (PS) II herbicide resistance occurs from mutations withinthe psbA gene, which enclosed the Q_(B) protein and is highly conservedamong many plants. Resistant plants possess mutations that alter aminoacids at specific positions within the Q_(B) proteins, e.g., residues219, 251, 255, 264, and 275 (Hirschberg and McIntosh. 1993; Galloway andMets. 1984; Gloden and Haselkorn. 1985; Erickson et al. 1984;Johanningmeier et al. 1987). Genes possessing these mutations can thusbe utilized to confer resistance to herbicides that function byinhibiting electron transport carried out by the PS II system.

Examples of these herbicides include dichlorophenyldimethylurea (DCMU),atrazine, metribuzine, lenacil, phenmedipham, loxynil and dinoseb.

The mutant psbA gene containing a serine to glycine mutation at residue264 is isolated from genomic DNA of Chlamydomonas using the appropriaterestriction endonucleases. The resulting fragment can be ligated intothe universal chloroplast expression vector pSBL-ctV2 and introducedinto E. coli XL1Blue. Purified plasmid from this E. coli strain isutilized to transform plants. Daniell (1997). Incorporation of themutant psbA genes into the chloroplast genome and selection of theappropriate transformants are carried out as previously described.Transformed plants producing the mutated psbA protein containing theserine to glycine substitution are resistant to Atrazine™ whereascontrol plants are not.

The mutant psbA gene containing a valine to isoleucine mutation atresidue 219 is isolated from genomic DNA of Chlamydomonas using theappropriate restriction endonucleases. A universal vector is constructedas described above. Transgenic plants like corn expressing psbAcontaining the valine to isoleucine mutation at residue 219 are expectedto be resistant to DCMU sprays.

EXAMPLE 15

Tolerance to Auxin Analogs. 2,4-D. The universal chloroplast expressionvector psbL-ctV2 can be cleaved with XbaI and ligated with a DNAfragment containing a gene encoding monooxygenase. The resultingconstruct can be transformed into chloroplasts to generate transgenicplants that contain multiple copies of the monooxygenase gene. Theresulting plants expressing high levels of monooxygenase and areexpected to be tolerant to 2,4-D.

EXAMPLE 16

Chloroplast Transformation for Insect Resistance. Tobacco plants can betransformed with universal vector pSBL-CtVHBt (FIG. 8A) which containthe cryIIA gene and expresses the CryIIA protoxin, thereby conferringresistance to insects pests like of the family Pyralidoe, such as thetobacco hornworm. Even insects which have developed a resistance or areless susceptible to Bt toxin are killed by the Bt toxin expressed by thegene in the chloroplast vector described herein.

Vector pSBL-CtVHBt is constructed by cleaving pSBL-CtVH with SmaI andligating the product with the cryIIA gene encoding the CryIIA protein.The product contained the cryIIA gene in the correct transcriptionalorientation (FIG. 8A)

Integration of the cryIIA gene into the tobacco chloroplast genome hasbeen confirmed by PCR analysis. Copy number of cryIIA per cell wasestimated to be 10,000 by performing Southern blots. The CryIIA proteinwas estimated to be between 5 and 10% of total cellular protein byperforming Western blots. This is the highest level of CryIIA proteinever reported in Bt transgenic plants. Excised leaf bioassays wereperformed using non-transformed “Petit Havana” and cryIIA-transformedtobacco. Five to ten larvae were placed on each leaf and evaluated formortality and leaf damage after 3-5 days. Using susceptible H. virescens(YDK), all larvae died within 3 days on cryII-A-transformed tobaccowhereas there was no mortality and essentially 100% defoliation on thenon-transformed “Petit Havana”. Similar results were obtained usingCryIAc-resistant (YHD2, 40-50,000 fold resistant) and CryII-A-resistant(CXC, 2000 fold resistant) H. virescens. In addition 100% mortality wasobserved against Helicoverpa zea (cotton bollworm), and Spodopteraexigua (beet armyworm), neither of which has been previously shown to bekilled by any Cry protein.

FIGS. 28A and 28B show a bioassay of (A) Control untransformed plant and(B) transgenic plant. Insects tested, demonstrated 100% mortality:Tobacco budworm susceptible to CryI, cotton bollworm and beet armywormresistant to CryI and CryII.

FIG. 29 shows total protein isolated by Western blot analysis fromControl (lane C) and transgenic plants (lane B). Lanes D-H representdifferent concentrations of purified CryIIA protein (1-20%). Lane Ashows protein standards.

Other controllable insects are described earlier in the description ofthe invention.

As will be apparent to those skilled in the art, in light of theforegoing description, many modifications, alterations, andsubstitutions are possible in the practice of the invention withoutdeparting from the spirit or scope thereof. It is intended that suchmodifications, alterations, and substitutions be included in the scopeof the claims.

All references cited in this text are expressly incorporated herein byreference.

REFERENCES

-   Arntzen Ph.D., Charles J. (1997) Public Health Reports 112: 190-197.-   Brixey, P. J., Guda, H. and Daniell, H. (1997) Biotechnol. Lett. 19,    395-399.-   Carlson, P. S. (1973) Proc. Natl. Acad. Sci. USA 70:598-602.-   Chaleff and Ray. (1984) Science 223:1148.-   Comai, L., Faciotti, D., Hiatt, W., Thomson, G., Rose, R., and    Stalker, D. (1983) Science 221:370.-   Daniell, H., Guda, C., McPherson, D. T., Xu, J., Zhang, X. and    Urry, D. W. (1997) Meth. Mol. Biol., 63:359-371.-   Daniell, H., and Guda, C. (1997) Chemistry and Industry, pages    555-558.-   Daniell, H., Krishnan, M. and McFadden, B. A. (1991) Plant Cell Rep.    9: 615-619.-   Daniell, H., and McFadden. (1987) Proc. Nat. Acad. Sci. (USA) 84:    6349-6353.-   Daniell, H., Vivekananda, J., Neilson, B., Ye, G. N., Tewari, K. K.,    and Sanford, J. C. (1990) Proc. Nat. Acad. Sci. (USA) 87: 88-92.-   Daniell, H. Porobo Dessai, A., Prakash, C. S. and Moar, W. J. (1994)    NATO Asi Series. Ed., J. H. Cherry. H86: 598-604.-   Darkocsik, C., Donovan, W. P. and Jany, C. S. (1990) Mol. Microbiol.    4: 2087-2094.-   Daniell, H., (1995) Inform. 6: 1365-1370.-   Daniell, H., Ramanujan, P., Krishnan, M., Gnanam, A. and    Rebeiz, C. A. (1983) Biochem. Biophys. Res. Comun 111:740-749.-   Daniell, H. and Rebeiz, C. A. (1982) Biochem. Biophys. Res. Comun.    106:466-471.-   Daniell, H. (1993) Methods in Enzymology. 217:536-556.-   Daniell, H. (1997a) Meth. Mol. Biol. 62: 453-488.-   DeBlock, M., Botterman, J., Vandewiele, M., Docky, J., Thuen, C.,    Gossele, V., Movva, N. R., Thomson, C., Van Montagu, M., and    Leemans, J. (1987) EMBO J. 6: 2513-2518.-   Erickson et al. (1984) Proc. Nat. Acad. Sci. (USA) 81:3617.-   Falco and Dumas. (1985) Genetics 109: 21.-   Gabard, J. M., Charest, P. J., Iyer, V. N. and Miki, B. L. (1989)    Plant Phys. 91:574-580.-   Galloway and Mets. (1984) Plant Physiol. 74: 469.-   Gloden and Haselkorn. (1985) Science 229: 1104.-   Guda, C., Zhang, X., McPherson, D. T., Xu, J., Cherry, J.,    Urry, D. W. and Daniell, H. (1995) Biotechnol. Lett. 17: 745-750.-   Hirschberg and McIntosh. (1993) Science 222: 1346.-   Johanningmeier et al. (1987) FEBS Lett 211: 221.-   Kanyand et al. (1994) Plant Cell Reports 14: 1-5.-   Kin Ying et al. (1996) The Plant Journal 10: 737-743.-   King, J. (1996) Science 274: 180-181.-   Laemmli, U. K. (1970) Nature 227: 680-685.-   Langevin, S. A., Clay, K. and Grace, J. B. (1990) Evolution 44:    1000-1008.-   Lewellyn and Fitt (1996) Molecular Breeding 2: 157-166.-   Lu, Z., Kunnimalaiyaan, M. and Nielsen, B. L. (1996) Plant Mol.    Biol. 32: 693-706.-   Lyons, P. C., May, G. D., Mason, H. S., and Arntzen, C. J., (1996)    Pharmaceutical News 3: 7-12.-   Maier, R. M., Neckerman, K., Igloi, G. L. and Kössel, H. (1995) J.    Mol. Biol. 251: 614-628.-   May, G. D., Mason, H. S., Lyons, P. C. (1996) American Chemical    Society, pp. 194-204.-   Miele, L. (1997) Elsevier Trends Journals, Vol. 15.-   Mikkelson, T. R., Anderson, B. and Jörgenson, R. B. (1996) Nature    380: 31.-   Miki, B. I., Labbe, H., Hatori, J., Ouellet, T., gabard, J.,    Sunohara, G., Charest, P. J. and Iyer, V. N. (1990) Theoretical    Applied Genetics 80: 449-458.-   McBride, K. E., Svab, Z., Schaaf, D. J., Hogan, P. S.,    Stalker, D. M. and Maliga, P. (1995) Bio/Technology 13: 362-365.-   Nielsen, B. L., Lu, Z. and Tewari, K. K. (1993) Plasmid 30: 197-211.-   Oard, J. H., Linscombe, S. D., Braveramn, M. P., Jodari, F.,    Blouin, D. C., Leech, M., Kohli, A., Vain, P. Cooley, J. C. and    Christou, P. (1996) Mol. Breed. 2: 359-368.-   Penazloza, V., et al. (1995) Plant Cell Reports 14:482-487.-   Rudraswamy, V., and Reichert, N. A. (1997) M. S. Thesis. Mississippi    State Univ.-   Rogers, S. O., and Bendich, A. J. (1988) in Plant Molecular Biology    Manual, ed. Gelvin, S. B. and Schilperoot, R. A. (Kulwer Academic    Publishers, Dordrecht, Netherlands) pp. A6:1-10.-   Sambrook, J., Fritch, E. F. and Maniatis, T. (1989) in Molecular    cloning. Cold Spring Harbor Press, Cold Spring Harbor, N.Y.-   Sanford, J. C. (1988) Trends In Biotech. 6: 299-302.-   Sankula, S., Braverman, M. P., Jordari, F., Linscombe, S. D. and    Oard, J. A. (1996) Weed Technol. 11: 70-75.-   Schultz, A., Wengenmayer, F. and Goodman, H. (1990) Critical Review    in Plant Sciences 9: 1-15.-   Shaner and Anderson, P. C., (1985), Biotechnology in Plant Science,    287.-   Sijmons, P. C., Cekker, B. M. M., Schrammeijer, B., Verwoerd, T. C.,    van den Elzen, P. J. M., Hoekema, A. (1990) Biotechnology 8:    217-221.-   Stalker, et al. (1985) J. Biol. Chem. 260: 4724.-   Stummann et al. (1988) Physiologia Plantarum 72:139-146.-   Svab, Z. and Maliga, P. (1993) Proc. Natl. Acad. Sci. (USA) 90:    913-917.-   Svab, Z., Hajdukiewicz, P. and Maglia, P. (1990) Proc. Nat. Acad.    Sci. (USA) 87: 8526-8530.-   Umbeck, P. F., et al. (1991) Econ. Entomology 84: 1943-1950.-   Urry, D. W., Nicol, A., Gowda, D. C., Hoban, L. D., McKee, A.,    Williams, T., Olsen, D. B. and Cox, B. A. (1993) in Biotechnological    Polymers Medical. Pharmaceutical and Industrial Applications, ed.    Gebelein, C. G. (Technomic Publishing Co., Inc., Atlanta, Ga.), pp.    82-103.-   Widner, W. R. and Whiteley, H. R. (1989) J. Bacteriol. 171: 961-974.-   Yadav, et al. (1986) Proc. Natl. Acad. (USA) 83: 4418-4422.-   Ye, G. N., Daniell, H. and Sanford, J. C. (1990) Plant Mol. Biol.    15: 809-819.-   Yeh, H., Ornstein-Goldstein, N., Indik, Z., Sheppard, P., Anderson,    N., Rosenbloom, J., Cicilia, G., Yoon, K. and Rosenbloom, J. (1987)    Collagen Related Res. 7: 235-247.-   Zhang, X., Guda, C., Datta, R., Dute, R., Urry, D. W. and    Daniell, H. (1996) Plant Cell Reports 15: 381-385.-   Zhang, X., Guda, C., Datta, R., Dute, R., Urry, D. W.,    Danell, H. (1995) Biotechnology Letters 17: 1279-1284.-   Zhang, X., Urry, D. W. and Daniell, H. (1996) Plant Cell Rep. 16:    174-179.-   Current Protocols in Molecular Biology, Asubel et al., eds., John    Wiley and Sons, Inc. (1997) Vol. I, II and III.-   Herbicide Resistance Crops, Agricultural, Environmental, Economic,    Regulatory and Technical Aspects, Duke, S. O., edt., CRC Press, Inc.    (1996).-   Herbicide Resistance in Plants, Biology and Biochemistry, Powles, S.    B., and Holtum, J. A. M., eds., CRC, Press, Inc. (1994.

1. A stably transformed herbicide resistant target plant species, or theprogeny thereof, which comprises a chloroplast stably transformed with auniversal integration and expression vector competent for stabletransformation of the chloroplast of different plant species, whichvector comprises an expression cassette which encodes a heterologousprotein of interest expressed by a heterologous DNA sequence in thechloroplast genome of the target plant species, which heterologous DNAconfers tolerance to said herbicide, and flanking each side of theexpression cassette, flanking DNA sequences derived from atranscriptionally active spacer region of a chloroplast genome, wherebystable integration of the heterologous DNA encoding the heterologousprotein into a transcriptionally active spacer region of the chloroplastgenome of the target plant was facilitated through homologousrecombination of the flanking sequences with the homologous sequences inthe target chloroplast genome.
 2. The herbicide resistant target plantof claim 1 in which the protein of interest is a mutant form of anenzyme which has decreased affinity for the herbicide than does thenaturally occurring enzyme.
 3. The herbicide resistant target plant ofclaim 1 wherein the herbicide is glyphosate.
 4. The herbicide resistanttarget plant of claim 3 wherein the enzyme is EPSP synthase.
 5. Theherbicide resistant target plant of claim 2 wherein the herbicide isselected from at least one of the following types: PSI, PSII, APP, auxinanalog, mitotic, tertiary amino methyl phosphoric acids type and ALSinhibiting types.
 6. The herbicide resistant target plant of claim 5wherein the herbicide is the PSI type selected from paraquat and diquat.7. The herbicide resistant target plant of claim 5 wherein the herbicideis selected from atrazine, dinoseb, lenacil and metribuzine.
 8. Theherbicide resistant target plant of claim 5 wherein the herbicide is ofthe APP type selected from cyclohexanedione, haloxyfop, clethodim andphenoxaprop, and the lower alkyl-substituted compound thereof.
 9. Theherbicide resistant target plant of claim 5 wherein the herbicide is anauxin analog selected from MCPA and 2,4-D.
 10. The herbicide resistanttarget plant of claim 5 wherein the herbicide is a mitotic typeherbicide, which is dinitroahiline.
 11. The herbicide resistant targetplant of claim 2 wherein the herbicide is a tertiary amino methylphosphoric acid type, which is glyphosate.
 12. The herbicide resistanttarget plant of claim 5 wherein the herbicide is an ALS inhibiting typeselected from sulfonylureas and imidazolines.
 13. The herbicideresistant target plant of claim 5 wherein the herbicide is selected frombromoxynil, methyl sulfuron, chlorsulfuron, phosphinothricin andimazapyr.
 14. The herbicide resistant target plant of claim 5 which ismaize, rice, grass, rye, barley, oat, wheat, soybean, peanut, grape,potato, sweet potato, pea, canola, tobacco, tomato or cotton.
 15. Theherbicide resistant target plant of claim 14 which is a homoplasmicplant.
 16. A process for conferring herbicide resistance to a targetplant species, which process comprises introducing into a plant cell ofthe target plant an integration and expression vector which vectorcomprises an expression cassette that comprises, operably joined, aheterologous DNA sequence coding for a protein of interest which confersresistance to a herbicide and control sequences positioned upstream fromthe 5′ and downstream from the 3′ ends of the coding sequence to provideexpression of the coding sequence in the chloroplast of the targetplant, a heterologous nucleotide sequence that confers a selectablephenotype other than tolerance to said herbicide, and flanking each sideof the expression cassette, flanking sequences which are derived from atranscriptionally active spacer region of which are homologous to atranscriptionally active spacer sequence of the target chloroplastgenome of the target plant, whereby stable integration of theheterologous coding sequence into a transcriptionally active spacerregion of the chloroplast genome of the target plant is facilitatedthrough homologous recombination of the flanking sequences with thehomologous sequences in the target chloroplast genome; and growing thetransformed plant.
 17. The process of claim 16 wherein the DNA sequencecodes for a mutant form of an enzyme which has decreased affinity forthe herbicide than does the naturally occurring enzyme.
 18. The processof claim 17 wherein the enzyme is EPSP synthase and the herbicide isglyphosate.
 19. The process of claim 18 wherein the DNA sequence is theEPSP synthase gene which is a mutant EPSP synthase gene.
 20. The processof claim 16 which comprises selecting the viable, transformed targetplants on a medium which is lethal to non-transformed plants.
 21. Theprocess of claim 20 wherein the viable transformed target plants arehomoplasmic plants.
 22. The process of claim 20 wherein the viabletransformed target plants are heteroplasmic plants.
 23. The process ofclaim 16 wherein the herbicide resistant target plant is tobacco, andthe nucleotide sequence confers a selectable phenotype which is lethalto tobacco or confers a visual trait that permits selection of thetransformed tobacco plants from the non-transformed plants.
 24. Theprocess of claim 23 wherein the lethal selectable phenotype isresistance to hygromycin to which tobacco is not naturally resistant.25. The process of claim 23 wherein the visual trait is the expressionof a color.
 26. The process of claim 16 for stably transforming thephenotype of a target plant species, whereby the grown transformed plantexpresses the selectable phenotype and another trait in addition to theexpression of that phenotype.
 27. The process of claim 26 wherein theselected phenotype is conferred by expression of the hygromycinβ-phosphotransferase gene and the additional trait that is conferred isresistance to the herbicide glyphosate.
 28. A process of determiningchloroplast transformation and expression of a target trait on the basisof a target plant species' acquisition of resistance to a selectedherbicide due to the transformation of the plant species, which processcomprises introducing into the plant a universal integration, andexpression vector competent for stably transforming the chloroplast ofdifferent plant species, which vector comprises an expression cassettewhich comprises, operably joined, a heterologous DNA sequence coding fora protein conferring the desired target trait and control sequencespositioned upstream from the 5′ and downstream from the 3′ ends of thecoding sequence to provide expression of the coding sequence in thechloroplast of a target plant, a heterologous nucleotide sequenceconferring herbicide resistance and flanking each side of the expressioncassette, flanking DNA sequences which comprise each one a portion ofthe intergenic spacer 2 region between the tRNA^(Ile) and the tRNA^(Ala)genes of the chloroplast genome, which are homologous to a spacersequence of the target chloroplast genome, which spacer sequence isconserved in the chloroplast genome of different plant species, wherebystable integration of the heterologous coding sequence into thechloroplast genome of the target plant is facilitated through homologousrecombination of the flanking sequences with the homologous sequences inthe target chloroplast genome; and exposing the plants into which thevector has been introduced to a lethal concentration of the herbicideand selecting the plants which do not die from exposure thereto, therebyhaving selected the transformed plants which express the desired targettrait.
 29. The process of claim 28 wherein the selected herbicide isselected from at least one of the following types: PSI, PSII, APP, auxinanalog, mitotic, tertiary amino methyl phosphoric acids and ALSinhibiting types.